Bacillus thuringiensis CryET29 compositions toxic to coleopteran insects and Ctenocephalides SPP.

ABSTRACT

Disclosed is a novel δ-endotoxin, designated CryET29, that exhibits insecticidal activity against siphonapteran insects, including larvae of the cat flea ( Ctenocephalides felis ), as well as against coleopteran insects, including the southern corn rootworm ( Diabrotica undecimpunctata ), western corn rootworm ( D. virgifera ), Colorado potato beetle ( Leptinotarsa decemlineata ), Japanese beetle ( Popillia japonica ), and red flour beetle ( Tribolium castaneum ). Also disclosed are nucleic acid segments encoding CryET29, recombinant vectors, host cells, and transgenic plants comprising a cryET29 DNA segment. Methods for making and using the disclosed protein and nucleic acid segments are disclosed as well as assays and diagnostic kits for detecting cryET29 and CryET29 sequences in vivo and in vitro.

1. BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates generally to the fields of molecularbiology. More particularly, certain embodiments concern methods andcompositions comprising DNA segments, and proteins derived frombacterial species. More particularly, it concerns a novel cryET29 genefrom Bacillus thuringiensis encoding a coleopteran- and cat flea-toxiccrystal protein. Various methods for making and using these DNAsegments, DNA segments encoding synthetically-modified CryET29 proteins,and native and synthetic crystal proteins are disclosed, such as, forexample, the use of DNA segments as diagnostic probes and templates forprotein production, and the use of proteins, fusion protein carriers andpeptides in various immunological and diagnostic applications. Alsodisclosed are methods of making and using nucleic acid segments in thedevelopment of transgenic plant cells containing the DNA segmentsdisclosed herein.

1.2 Description of the Related Art

1.2.1 Bacillus thuringiensis Crystal Proteins

Bacillus thuringiensis is a Gram-positive bacterium that producesδ-endotoxins known as crystal proteins which are specifically toxic tocertain orders and species of insects. Many different strains of B.thuringiensis have been shown to produce insecticidal crystal proteins.Compositions including B. thuringiensis strains which produceinsecticidal proteins have been commercially available and used asenvironmentally acceptable insecticides because they are quite toxic tothe specific target insect, but are harmless to plants and othernon-targeted organisms.

The B. thuringiensis crystal protein is toxic in the insect only afteringestion when the alkaline pH and proteolytic enzymes in the insectmid-gut solubilize the crystal protein and release the toxic components.These components disrupt the mid-gut cells causing the insect to ceasefeeding and, eventually to die. In fact, B. thuringiensis has proven tobe an effective and environmentally safe insecticide in dealing withvarious insect pests.

As noted by Hofte et al., (1989) the majority of insecticidal B.thuringiensis strains are active against insect of the orderLepidoptera, i.e., caterpillar insects. Other B. thuringiensis strainsare insecticidally active against insects of the order Diptera, i.e.,flies and mosquitoes, or against both lepidopteran and dipteran insects.In recent years, a few B. thuringiensis strains have been reported asproducing crystal proteins that are toxic to insects of the orderColeoptera, i.e., beetles. To date, there have been no reports of B.thuringiensis strains active on fleas of the Genus, Ctenocephalides, inthe order Siphonaptera.

The dipteran-active Cyt toxins differ from most of the other B.thuringiensis insecticidal crystal proteins in that they are smaller anddo not share conserved blocks of sequence homology. These proteinsdemonstrate broad cytolytic activity in vitro, yet are specificallytoxic to larvae of dipteran insects in vivo. These properties have beendescribed elsewhere (Chilcott and Ellar, 1988).

1.2.2 Genetics of Crystal Proteins

A number of genes encoding crystal proteins have been cloned fromseveral strains of B. thuringiensis. The review by Höfte et al. (1989)discusses the genes and proteins that were identified in B.thuringiensis prior to 1990, and sets forth the nomenclature andclassification scheme which has traditionally been applied to B.thuringiensis genes and proteins. cryI genes encode lepidopteran-toxicCryI proteins. cryII genes encode CryII proteins that are toxic to bothlepidopterans and dipterans. cryIII genes encode coleopteran-toxicCryIII proteins, while cryIV genes encode dipteran-toxic CryIV proteins.

Recently a new nomenclature has been proposed which systematicallyclassifies the cry genes based upon DNA sequence homology rather thanupon insect specificities. This classification scheme is shown inTable 1. TABLE 1 Revised B. thuringiensis δ-Endotoxin GeneNomenclature^(a) New Old GenBank Accession # Cry1Aa CryIA(a) M11250Cry1Ab CryIA(b) M13898 Cry1Ac CryIA(c) M11068 Cry1Ad CryIA(d) M73250Cry1Ae CryIA(e) M65252 Cry1Ba CryIB X06711 Cry1Bb ET5 L32020 Cry1Bc PEG5Z46442 Cry1Ca CryIC X07518 Cry1Cb CryIC(b) M97880 Cry1Da CryID X54160Cry1Db PrtB Z22511 Cry1Ea CryIE X53985 Cry1Eb CryIE(b) M73253 Cry1FaCryIF M63897 Cry1Fb PrtD Z22512 Cry1G PrtA Z22510 Cry1H PrtC Z22513Cry1Hb U35780 Cry1Ia CryV X62821 Cry1Ib CryV U07642 Cry1Ja ET4 L32019Cry1Jb ET1 U31527 Cry1K U28801 Cry2Aa CryIIA M31738 Cry2Ab CryIIB M23724Cry2Ac CryIIC X57252 Cry3A CryIIIA M22472 Cry3Ba CryIIIB X17123 Cry3BbCryIIIB2 M89794 Cry3C CryIIID X59797 Cry4A CryIVA Y00423 Cry4B CryIVBX07423 Cry5Aa CryVA(a) L07025 Cry5Ab CryVA(b) L07026 Cry5B U19725 Cry6ACryVIA L07022 Cry6B CryVIB L07024 Cry7Aa CryIIIC M64478 Cry7Ab CryIIICbU04367 Cry8A CryIIIE U04364 Cry8B CryIIIG U04365 Cry8C CryIIIF U04366Cry9A CryIG X58120 Cry9B CryIX X75019 Cry9C CryIH Z37527 Cry10A CryIVCM12662 Cry11A CryIVD M31737 Cry11B Jeg80 X86902 Cry12A CryVB L07027Cry13A CryVC L07023 Cry14A CryVD U13955 Cry15A 34 kDa M76442 Cry16Acbm71 X94146 Cyt1A CytA X03182 Cyt2A CytB Z14147 To Be Assigned CryET29,Present Invention To Be Assigned^(a)Adapted from: http://www.susx.ac.uk:80/users/bafn6/bt/index.html1.2.3 Identification of Crystal Proteins Toxic To Coleopteran Insects

The cloning and expression of a gene encoding a 26-kDa mosquitocidaltoxin from the dipteran-active B. thuringiensis var. israelensis hasbeen described (Ward et al., 1984), and the nucleotide sequence of thisgene was reported (Ward and Ellar, 1986). The molecular mass of thetoxin protein, CytA, calculated from the deduced amino acid sequence wasdetermined to be 27,340 Da.

The nucleotide sequence of the gene for a 27-kDa mosquitocidal Cytprotein isolated from B. thuringiensis var. morrisoni strain PG14 hasbeen disclosed (Earp and Ellar, 1987). The sequence of this toxinprotein was found to differ by only one amino acid residue from theCytIA protein of B. thuringiensis var. israelensis.

The identification of a 25-kDa protein that exhibits cytolytic activityin vitro when activated by proteolysis from the mosquitocidal B.thuringiensis var. kyushuensis was described earlier (Knowles et al.,1992), and the nucleotide sequence of the gene for this protein, CytB,was reported (Koni and Ellar, 1993). The predicted molecular mass of theCytB protein is 29,236 Da and the deduced amino acid sequence is quitedistinct, although it does share significant sequence similarity withthe CytA protein of B. thuringiensis var. israelensis.

The cloning and characterization of the gene for a 30-kDa toxin proteinwith activity on coleopteran and dipteran insects has been described(Intl. Pat. Appl. Pub. No. WO 95/02693, 1995). This gene, isolated fromB. thuringiensis PS201T6, encodes a protein of 29,906 Da which exhibitsa 64% sequence identity with the CytA toxin of B. thuringiensis var.israelensis.

2. SUMMARY OF THE INVENTION

The present invention provides a novel B. thuringiensis insecticidalcrystal protein (designated CryET29) and the gene which encodes it(designated cryET29) which contain amino acid and nucleic acidsequences, respectively, showing little homology to the δ-endotoxinproteins and genes of the prior art. Surprisingly, the CryET29 proteinof the present invention demonstrates remarkable insecticidal activityagainst not only insects of the order Coleoptera, but also againstfleas, and in particular larvae of the cat flea, Ctenocephalides felis.

In one important embodiment, the invention provides an isolated andpurified amino acid segment comprising a B. thuringiensis CryET29insecticidal crystal protein (SEQ ID NO:2) comprising the amino acidsequence illustrated in FIG. 1A and FIG. 1B. The coding region for theCryET29 protein is from nucleotide 29 to 721 of SEQ ID NO: 1. TheCryET29 protein exhibits insecticidal activity against Coleopterans suchas the southern corn rootworm, western corn rootworm, Colorado potatobeetle, Japanese beetle, and red flour beetle. In related embodiments,methods for making and using this protein, derivatives and mutantsthereof, and antibodies directed against these proteins are alsodisclosed.

In another important embodiment, the invention provides an isolated andpurified nucleic acid segment comprising the cryET29 gene which encodesthe CryET29 crystal protein disclosed herein. The nucleotide sequence ofthe cryET29 gene is given in SEQ ID NO:1 and illustrated in FIG. 1A andFIG. 1B. In related embodiments, methods for making, using, altering,mutagenizing, assaying, and quantitating these nucleic acid segments arealso disclosed. Also disclosed are diagnostic methods and assay kits forthe identification and detection of related cry gene sequences in avariety of in vitro and in vivo methodologies.

Another aspect of the present invention is a Bacillus thuringiensis cellthat produces a CryET29 crystal protein. In a preferred embodiment, thecell is a Bacillus thuringiensis bacterial strain designated B.thuringiensis EG4096 which has been deposited with the AgriculturalResearch Culture Collection, Northern Regional Research Laboratory(NRRL), on May 30, 1996 and assigned the Accession No. NRRL B-21582. B.thuringiensis EG4096, further described in Examples 1, 2, and 3, is anaturally-occurring bacterium that comprises a cryET29 gene (SEQ IDNO: 1) of the present invention. EG4096 produces a novel insecticidalcrystal protein of approximately 26-kDa, which the inventors havedesignated CryET29 (SEQ ID NO:2). Most preferably, the Bacillusthuringiensis cell has the NRRL accession number NRRL B-21582.

A further aspect of the present invention is a plasmid, cosmid, orvector that comprises the nucleic acid sequence of a whole or a portionof the cryET29 gene (SEQ NO ID: 1), a transformed host cell comprising anative or recombinant cryET29 gene, a culture of a recombinant bacteriumtransformed with such plasmid, the bacterium preferably being B.thuringiensis such as the recombinant strains EG11494 and EG11502,described in Example 7, and most preferably a biologically-pure cultureof such a bacterial strain. EG11494 was deposited on May 30, 1996 underthe terms of the Budapest Treaty with the NRRL and given the Accessionnumber NRRL B-21583. Alternatively, the E. coli recombinant strainsEG11513 and EG1514 comprising the novel cryET29 gene, are also preferredhosts for expression of the CryET29 protein.

2.1 cryET29 DNA Segments

The present invention also concerns DNA segments, that can be isolatedfrom virtually any source, that are free from total genomic DNA and thatencode the whole or a portion of the novel peptides disclosed herein.The cryET29 gene (SEQ ID NO: 1; FIG. 1A and FIG. 1B) encodes the 26-kDaCryET29 protein having an amino acid sequence shown in FIG. 1A and FIG.1B (SEQ ID NO:2). DNA segments encoding these peptide species may proveto encode proteins, polypeptides, subunits, functional domains, and thelike of crystal protein-related or other non related gene products. Inaddition these DNA segments may be synthesized entirely in vitro usingmethods that are well-known to those of skill in the art.

As used herein, the term “DNA segment” refers to a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species.Therefore, a DNA segment encoding a crystal protein or peptide refers toa DNA segment that contains crystal protein coding sequences yet isisolated away from, or purified free from, total genomic DNA of thespecies from which the DNA segment is obtained, which in the instantcase is the genome of the Gram-positive bacterial genus, Bacillus, andin particular, the species known as B. thuringiensis. Included withinthe term “DNA segment”, are DNA segments and smaller fragments of suchsegments, and also recombinant vectors, including, for example,plasmids, cosmids, phagemids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified crystalprotein-encoding gene refers to a DNA segment which may include inaddition to peptide encoding sequences, certain other elements such as,regulatory sequences, isolated substantially away from other naturallyoccurring genes or protein-encoding sequences. In this respect, the term“gene” is used for simplicity to refer to a functional protein-,polypeptide- or peptide-encoding unit. As will be understood by those inthe art, this functional term includes not only genomic sequences,including extrachromosomal DNA sequences, but also operon sequencesand/or engineered gene segments that express, or may be adapted toexpress, proteins, polypeptides or peptides. “Isolated substantiallyaway from other coding sequences” means that the gene of interest, inthis case, a gene encoding a bacterial crystal protein, forms thesignificant part of the coding region of the DNA segment, and that theDNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or operon coding regions. Of course, this refers to the DNAsegment as originally isolated, and does not exclude genes, recombinantgenes, synthetic linkers, or coding regions later added to the segmentby the hand of man.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences that encode a Cryprotein or peptide species that includes within its amino acid sequencean amino acid sequence essentially as set forth in SEQ ID NO:2. Morepreferably, the DNA sequence comprises a nucleic acid sequence thatencodes a Cry protein or peptide species that includes within its aminoacid sequence an at least ten amino acid contiguous sequence of SEQ IDNO:2.

The term “a sequence essentially as set forth in SEQ ID NO:2,” meansthat the sequence substantially corresponds to a portion of the sequenceof SEQ ID NO:2 and has relatively few amino acids that are not identicalto, or a biologically functional equivalent of, the amino acids of anyof these sequences. The term “biologically functional equivalent” iswell understood in the art and is further defined in detail herein(e.g., see Illustrative Embodiments). Accordingly, sequences that havebetween about 70% and about 80%, or more preferably between about 81 %and about 90%, or even more preferably between about 91% and about 99%amino acid sequence identity or functional equivalence to the aminoacids of SEQ ID NO:2 will be sequences that are “essentially as setforth in SEQ ID NO:2.”

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared thatinclude a short contiguous stretch encoding the whole or a portion ofthe peptide sequence disclosed in SEQ ID NO:2, or that are identical toor complementary to DNA sequences which encode the peptide disclosed inSEQ ID NO:2, and particularly the DNA segment disclosed in SEQ ID NO: 1.For example, DNA sequences such as about 14 nucleotides, and that are upto about 10,000, about 5,000, about 3,000, about 2,000, about 1,000,about 500, about 200, about 100, about 50, and about 14 base pairs inlength (including all intermediate lengths) are also contemplated to beuseful.

It will be readily understood that “intermediate lengths”, in thesecontexts, means any length between the quoted ranges, such as 14, 15,16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51,52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.;including all integers through the 200-500; 500-1,000; 1,000-2,000;2,000-3,000; 3,000-5,000; and up to and including sequences of about10,000 nucleotides and the like.

It will also be understood that this invention is not limited to theparticular nucleic acid sequences which encode peptides of the presentinvention, or which encode the amino acid sequence of SEQ ID NO:2,including the DNA sequence which is particularly disclosed in SEQ IDNO:1. Recombinant vectors and isolated DNA segments may thereforevariously include the peptide-coding regions themselves, coding regionsbearing selected alterations or modifications in the basic codingregion, or they may encode larger polypeptides that nevertheless includethese peptide-coding regions or may encode biologically functionalequivalent proteins or peptides that have variant amino acids sequences.

The DNA segments of the present invention encompassbiologically-functional, equivalent peptides. Such sequences may ariseas a consequence of codon redundancy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally-equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test mutants inorder to examine activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, e.g.,where the peptide-coding regions are aligned within the same expressionunit with other proteins or peptides having desired functions, such asfor purification or immunodetection purposes (e.g., proteins that may bepurified by affinity chromatography and enzyme label coding regions,respectively).

Recombinant vectors form further aspects of the present invention.Particularly useful vectors are contemplated to be those vectors inwhich the coding portion of the DNA segment, whether encoding a fulllength protein or smaller peptide, is positioned under the control of apromoter. The promoter may be in the form of the promoter that isnaturally associated with a gene encoding peptides of the presentinvention, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment or exon, for example, usingrecombinant cloning and/or PCR™ technology, in connection with thecompositions disclosed herein.

2.2 DNA Segments as Hybridization Probes and Primers

In addition to their use in directing the expression of crystal proteinsor peptides of the present invention, the nucleic acid sequencescontemplated herein also have a variety of other uses. For example, theyalso have utility as probes or primers in nucleic acid hybridizationembodiments. As such, it is contemplated that nucleic acid segments thatcomprise a sequence region that consists of at least a 14 nucleotidelong contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous DNA segment of SEQ IDNO: 1 will find particular utility. Longer contiguous identical orcomplementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200,500, 1000, 2000, 5000, 10000 etc. (including all intermediate lengthsand up to and including full-length sequences) will also be of use incertain embodiments.

The ability of such nucleic acid probes to specifically hybridize tocrystal protein-encoding sequences will enable them to be of use indetecting the presence of complementary sequences in a given sample.However, other uses are envisioned, including the use of the sequenceinformation for the preparation of mutant species primers, or primersfor use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200nucleotides or so, identical or complementary to the DNA sequence of SEQID NO: 1, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting. Smaller fragments willgenerally find use in hybridization embodiments, wherein the length ofthe contiguous complementary region may be varied, such as between about10-14 and about 100 or 200 nucleotides, but larger contiguouscomplementary stretches may be used, according to the lengthcomplementary sequences one wishes to detect.

The use of a hybridization probe of about 14 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 14 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 20 contiguous nucleotides,or even longer where desired.

Of course, fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. Nos. 4,683,195 and4,683,202 (each incorporated herein by reference), by introducingselected sequences into recombinant vectors for recombinant production,and by other recombinant DNA techniques generally known to those ofskill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.15 M NaCl at temperatures ofabout 50° C. to about 70° C. Such selective conditions tolerate little,if any, mismatch between the probe and the template or target strand,and would be particularly suitable for isolating crystalprotein-encoding DNA segments. Detection of DNA segments viahybridization is well-known to those of skill in the art, and theteachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (each incorporatedherein by reference) are exemplary of the methods of hybridizationanalyses. Teachings such as those found in the texts of Maloy et al.,1994; Segal 1976; Prokop, 1991; and Kuby, 1991, are particularlyrelevant.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate crystalprotein-encoding sequences from related species, functional equivalents,or the like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ conditions such as about 0.15 Mto about 0.9 M salt, at temperatures ranging from about 20° C. to about55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmental undesirable reagents. In the case of enzyme tags,calorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

2.3 Recombinant Vectors and Protein Expression

The invention also discloses and claims a composition comprising aCryET29 crystal protein. The composition may comprises bacterial hostcells which express a CryET29 crystal protein, inclusion bodies orcrystals containing the CryET29 protein, culture supernatant, disruptedcells, cell extracts, lysates, homogenates, and the like. Thecompositions may be in aqueous form, or alternatively, in dry, semi-wet,or similar forms such as cell paste, cell pellets, or alternativelyfreeze dried, powdered, lyophilized, evaporated, or otherwise similarlyprepared in dry form. Such means for preparing crystal proteins arewell-known to those of skill in the art of bacterial protein isolationand purification. In certain embodiments, the crystal proteins may bepurified, concentrated, admixed with other reagents, or processed to adesired final form. Preferably, the composition will comprise from about1% to about 90% by weight of the crystal protein, and more preferablyfrom about 5%, to about 50% by weight.

In a preferred embodiment, the crystal protein compositions of theinvention may be prepared by a process which comprises the steps ofculturing a Bacillus thuringiensis cell which expresses a CryET29crystal protein under conditions effective to produce such a protein,and then obtaining the protein from the cell. The obtaining of such acrystal protein may further include purifying, concentrating,processing, or admixing the protein with one or more reagents.Preferably, the CryET29 crystal protein is obtained in an amount of frombetween about 1% to about 90% by weight and more preferably from about5% to about 50% by weight.

The invention also relates to a method of preparing a CryET29 crystalprotein composition. Such a method generally involves the steps ofculturing a Bacillus thuringiensis cell which expresses a CryET29crystal protein under conditions effective to produce the protein, andthen obtaining the protein so produced. In a preferred embodiment theBacillus thuringiensis cell is an NRRL B-21582 cell, or any Bacillusthuringiensis cell which contains a cryET29gene segment. Alternatively,the recombinant plasmid vectors of the invention may be used totransform other suitable bacterial or eukaryotic cells to produce thecrystal protein of the invention. Prokaryotic host cells includingGram-negative cells such as E. coli, Pseudomonas spp. and relatedEnterobacteraceae, or Gram-positive cells such as Bacillus spp.(including B. megaterium, B. subtilis, and B. thuringiensis) and thelike are all contemplated to be useful in the preparation of the crystalproteins of the invention. Particularly preferred E. coli strainsinclude NRRL B-21624, deposited with the NRRL under the terms of theBudapest Treaty.

In such embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a DNA segment encoding a crystal protein orpeptide in its natural environment. Such promoters may include promotersnormally associated with other genes, and/or promoters isolated from anybacterial, viral, eukaryotic, or plant cell. Naturally, it will beimportant to employ a promoter that effectively directs the expressionof the DNA segment in the cell type, organism, or even animal, chosenfor expression. The use of promoter and cell type combinations forprotein expression is generally known to those of skill in the art ofmolecular biology, for example, see Sambrook et al., 1989. The promotersemployed may be constitutive, or inducible, and can be used under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins or peptides. Appropriate promoter systemscontemplated for use in high-level expression include, but are notlimited to, the Pichia expression vector system (Pharmacia LKBBiotechnology).

In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of crystal peptidesor epitopic core regions, such as may be used to generate anti-crystalprotein antibodies, also falls within the scope of the invention. DNAsegments that encode peptide antigens from about 8 to about 50 aminoacids in length, or more preferably, from about 8 to about 30 aminoacids in length, or even more preferably, from about 8 to about 20 aminoacids in length are contemplated to be particularly useful. Such peptideepitopes may be amino acid sequences which comprise contiguous aminoacid sequences from SEQ ID NO:2.

2.4 Crystal Protein Transgenes and Transgenic Host Cells

In yet another aspect, the present invention provides methods forproducing a transgenic cell, and in particular a plant or animal cellwhich expresses a nucleic acid segment encoding the novel CryET29crystal protein of the present invention. The process of producingtransgenic cells is well-known in the art. In general, the methodcomprises transforming a suitable host cell with a DNA segment whichcontains a promoter operatively linked to a coding region that encodes aB. thuringiensis CryET29 crystal protein. Such a coding region isgenerally operatively linked to a transcription-terminating region,whereby the promoter is capable of driving the transcription of thecoding region in the cell, and hence providing the cell the ability toproduce the recombinant protein in vivo. Alternatively, in instanceswhere it is desirable to control, regulate, or decrease the amount of aparticular recombinant crystal protein expressed in a particulartransgenic cell, the invention also provides for the expression ofcrystal protein antisense mRNA. The use of antisense mRNA as a means ofcontrolling or decreasing the amount of a given protein of interest in acell is well-known in the art.

In a preferred embodiment, the invention encompasses a plant cell whichhas been transformed with a nucleic acid segment of the invention, andwhich expresses a gene or gene segment encoding one or more of the novelpolypeptide compositions disclosed herein. As used herein, the term“transgenic plant cell” is intended to refer to a plant cell that hasincorporated DNA sequences, including but not limited to genes which areperhaps not normally present, DNA sequences not normally transcribedinto RNA or translated into a protein (“expressed”), or any other genesor DNA sequences which one desires to introduce into the non-transformedplant, such as genes which may normally be present in thenon-transformed plant but which one desires to either geneticallyengineer or to have altered expression.

It is contemplated that in some instances the genome of a transgenicplant of the present invention will have been augmented through thestable introduction of a cryET29 transgene, either native cryET29, orsynthetically modified or mutated cryET29. In some instances, more thanone transgene will be incorporated into the genome of the transformedhost plant cell. Such is the case when more than one crystalprotein-encoding DNA segment is incorporated into the genome of such aplant. In certain situations, it may be desirable to have one, two,three, four, or even more B. thuringiensis crystal proteins (eithernative or recombinantly-engineered) incorporated and stably expressed inthe transformed transgenic plant. In preferred embodiments, theintroduction of the transgene into the genome of the plant cell resultsin a stable integration wherein the offspring of such plants alsocontain a copy of the transgene in their genome. The inheritibility ofthis genetic element by the progeny of the plant into which the gene wasoriginally introduced is a preferred aspect of this invention.

A preferred gene which may be introduced includes, for example, acrystal protein-encoding a DNA sequence from bacterial origin, andparticularly one or more of those described herein which are obtainedfrom Bacillus spp. Highly preferred nucleic acid sequences are thoseobtained from B. thuringiensis, or any of those sequences which havebeen genetically engineered to decrease or increase the insecticidalactivity of the crystal protein in such a transformed host cell.

Means for transforming a plant cell and the preparation of a transgeniccell line are well-known in the art (as exemplified in U.S. Pat. Nos.5,550,318; 5,508,468; 5,482,852; 5,384,253; 5,276,269; and 5,225,341,all specifically incorporated herein by reference), and are brieflydiscussed herein. Vectors, plasmids, cosmids, YACs (yeast artificialchromosomes) and DNA segments for use in transforming such cells will,of course, generally comprise either the operons, genes, or gene-derivedsequences of the present invention, either native, orsynthetically-derived, and particularly those encoding the disclosedcrystal proteins. These DNA constructs can further include structuressuch as promoters, enhancers, polylinkers, or even gene sequences whichhave positively- or negatively-regulating activity upon the particulargenes of interest as desired. The DNA segment or gene may encode eithera native or modified crystal protein, which will be expressed in theresultant recombinant cells, and/or which will impart an improvedphenotype to the regenerated plant.

Such transgenic plants may be desirable for increasing the insecticidalresistance of a monocotyledonous or dicotyledonous plant, byincorporating into such a plant, a transgenic DNA segment encoding aCryET29 crystal protein which is toxic to coleopteran insects.Particularly preferred plants include corn, wheat, soybeans, turfgrasses, ornamental plants, fruit trees, shrubs, vegetables, grains,legumes, and the like, or any plant into which introduction of a crystalprotein transgene is desired.

In a related aspect, the present invention also encompasses a seedproduced by the transformed plant, a progeny from such seed, and a seedproduced by the progeny of the original transgenic plant, produced inaccordance with the above process. Such progeny and seeds will have acrystal protein transgene stably incorporated into its genome, and suchprogeny plants will inherit the traits afforded by the introduction of astable transgene in Mendelian fashion. All such transgenic plants havingincorporated into their genome transgenic DNA segments encoding aCryET29 crystal protein or polypeptide are aspects of this invention.

2.5 Site-Specific Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, for example, incorporating one or more of the foregoingconsiderations, by introducing one or more nucleotide sequence changesinto the DNA. Site-specific mutagenesis allows the production of mutantsthrough the use of specific oligonucleotide sequences which encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids are alsoroutinely employed in site directed mutagenesis which eliminates thestep of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.

2.6 Antibody Compositions and Methods of Producing

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which bind to the crystalproteins disclosed herein. Means for preparing and characterizingantibodies are well known in the art (See, e.g, Harlow and Lane, 1988;incorporated herein by reference). The methods for generating monoclonalantibodies (mAbs) generally begin along the same lines as those forpreparing polyclonal antibodies. Briefly, a polyclonal antibody isprepared by immunizing an animal with an immunogenic composition inaccordance with the present invention and collecting antisera from thatimmunized animal. A wide range of animal species can be used for theproduction of antisera. Typically the animal used for production ofanti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or agoat. Because of the relatively large blood volume of rabbits, a rabbitis a preferred choice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified crystal protein, polypeptide or peptide. Theimmunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep frog cells is alsopossible. The use of rats may provide certain advantages (Goding, 1986,pp. 60-61), but mice are preferred, with the BALB/c mouse being mostpreferred as this is most routinely used and generally gives a higherpercentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×108 lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, OF, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S 194/5XXO Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, (Gefter et aL, 1977). The use of electrically inducedfusion methods is also appropriate (Goding, 1986, pp. 71-74).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

2.7 Crystal Protein Screening and Immunodetection Kits

The present invention also provides compositions, methods and kits forscreening samples suspected of containing a CryET29 δ-endotoxin or agene encoding such a crystal protein. Such screening may be performed onsamples such as transformed host cells, transgenic plants, progeny orseed thereof, or laboratory samples suspected of containing or producingsuch a polypeptide or nucleic acid segment. A kit can contain a novelnucleic acid segment or an antibody of the present invention. The kitcan contain reagents for detecting an interaction between a sample and anucleic acid or an antibody of the present invention. The providedreagent can be radio-, fluorescently- or enzymatically-labeled. The kitcan contain a known radiolabeled agent capable of binding or interactingwith a nucleic acid or antibody of the present invention.

The reagent of the kit can be provided as a liquid solution, attached toa solid support or as a dried powder. Preferably, when the reagent isprovided in a liquid solution, the liquid solution is an aqueoussolution. Preferably, when the reagent provided is attached to a solidsupport, the solid support can be chromatograph media, a test platehaving a plurality of wells, or a microscope slide. When the reagentprovided is a dry powder, the powder can be reconstituted by theaddition of a suitable solvent, that may be provided.

In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that thecrystal proteins or peptides of the present invention may be employed todetect antibodies having reactivity therewith, or, alternatively,antibodies prepared in accordance with the present invention, may beemployed to detect crystal proteins or crystal protein-relatedepitope-containing peptides. In general, these methods will includefirst obtaining a sample suspected of containing such a protein, peptideor antibody, contacting the sample with an antibody or peptide inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of an immunocomplex, andthen detecting the presence of the immunocomplex.

In general, the detection of immunocomplex formation is quite well knownin the art and may be achieved through the application of numerousapproaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), indirectimmunofluorescence techniques and the like. Generally, immunocomplexformation will be detected through the use of a label, such as aradiolabel or an enzyme tag (such as alkaline phosphatase, horseradishperoxidase, or the like). Of course, one may find additional advantagesthrough the use of a secondary binding ligand such as a second antibodyor a biotin/avidin ligand binding arrangement, as is known in the art.

For assaying purposes, it is proposed that virtually any samplesuspected of comprising either a crystal protein or peptide or a crystalprotein-related peptide or antibody sought to be detected, as the casemay be, may be employed. It is contemplated that such embodiments mayhave application in the titering of antigen or antibody samples, in theselection of hybridomas, and the like. In related embodiments, thepresent invention contemplates the preparation of kits that may beemployed to detect the presence of crystal proteins or related peptidesand/or antibodies in a sample. Samples may include cells, cellsupernatants, cell suspensions, cell extracts, enzyme fractions, proteinextracts, or other cell-free compositions suspected of containingcrystal proteins or peptides. Generally speaking, kits in accordancewith the present invention will include a suitable crystal protein,peptide or an antibody directed against such a protein or peptide,together with an immunodetection reagent and a means for containing theantibody or antigen and reagent. The immunodetection reagent willtypically comprise a label associated with the antibody or antigen, orassociated with a secondary binding ligand. Exemplary ligands mightinclude a secondary antibody directed against the first antibody orantigen or a biotin or avidin (or streptavidin) ligand having anassociated label. Of course, as noted above, a number of exemplarylabels are known in the art and all such labels may be employed inconnection with the present invention.

The container will generally include a vial into which the antibody,antigen or detection reagent may be placed, and preferably suitablyaliquotted. The kits of the present invention will also typicallyinclude a means for containing the antibody, antigen, and reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow-molded plastic containers into which thedesired vials are retained.

2.8 ELISAs and Immunoprecipitation

ELISAs may be used in conjunction with the invention. In an ELISA assay,proteins or peptides incorporating crystal protein antigen sequences areimmobilized onto a selected surface, preferably a surface exhibiting aprotein affinity such as the wells of a polystyrene microtiter plate.After washing to remove incompletely adsorbed material, it is desirableto bind or coat the assay plate wells with a nonspecific protein that isknown to be antigenically neutral with regard to the test antisera suchas bovine serum albumin (BSA), casein or solutions of milk powder. Thisallows for blocking of nonspecific adsorption sites on the immobilizingsurface and thus reduces the background caused by nonspecific binding ofantisera onto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween®. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from about 2 to about 4 hours, at temperatures preferably on theorder of about 25° to about 27° C. Following incubation, theantisera-contacted surface is washed so as to remove non-immunocomplexedmaterial. A preferred washing procedure includes washing with a solutionsuch as PBS/Tween®, or borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound antigen, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for the first. To provide adetecting means, the second antibody will preferably have an associatedenzyme that will generate a color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact and incubate the antisera-bound surface with a urease orperoxidase-conjugated anti-human IgG for a period of time and underconditions which favor the development of immunocomplex formation (e.g.,incubation for 2 hours at room temperature in a PBS-containing solutionsuch as PBS Tween®).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2, 2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectra spectrophotometer.

The anti-crystal protein antibodies of the present invention areparticularly useful for the isolation of other crystal protein antigensby immunoprecipitation. Immunoprecipitation involves the separation ofthe target antigen component from a complex mixture, and is used todiscriminate or isolate minute amounts of protein. For the isolation ofmembrane proteins cells must be solubilized into detergent micelles.Nonionic salts are preferred, since other agents such as bile salts,precipitate at acid pH or in the presence of bivalent cations.

In an alternative embodiment the antibodies of the present invention areuseful for the close juxtaposition of two antigens. This is particularlyuseful for increasing the localized concentration of antigens, e.g.enzyme-substrate pairs.

2.9 Western Blots

The compositions of the present invention will find great use inimmunoblot or western blot analysis. The anti-peptide antibodies may beused as high-affinity primary reagents for the identification ofproteins immobilized onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. In conjunction withimmuno-precipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. This is especially useful when the antigens studied areimmunoglobulins (precluding the use of immunoglobulins binding bacterialcell wall components), the antigens studied cross-react with thedetecting agent, or they migrate at the same relative molecular weightas a cross-reacting signal.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

2.10 Epitopic Core Sequences

The present invention is also directed to protein or peptidecompositions, free from total cells and other peptides, which comprise apurified protein or peptide which incorporates an epitope that isimmunologically cross-reactive with one or more anti-crystal proteinantibodies. In particular, the invention concerns epitopic coresequences derived from Cry proteins or peptides.

As used herein, the term “incorporating an epitope(s) that isimmunologically cross-reactive with one or more anti-crystal proteinantibodies” is intended to refer to a peptide or protein antigen whichincludes a primary, secondary or tertiary structure similar to anepitope located within a crystal protein or polypeptide. The level ofsimilarity will generally be to such a degree that monoclonal orpolyclonal antibodies directed against the crystal protein orpolypeptide will also bind to, react with, or otherwise recognize, thecross-reactive peptide or protein antigen. Various immunoassay methodsmay be employed in conjunction with such antibodies, such as, forexample, Western blotting, ELISA, RIA, and the like, all of which areknown to those of skill in the art.

The identification of Cry immunodominant epitopes, and/or theirfunctional equivalents, suitable for use in vaccines is a relativelystraightforward matter. For example, one may employ the methods of Hopp,as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference,which teaches the identification and preparation of epitopes from aminoacid sequences on the basis of hydrophilicity. The methods described inseveral other papers, and software programs based thereon, can also beused to identify epitopic core sequences (see, e.g., Jameson and Wolf,1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acidsequence of these “epitopic core sequences” may then be readilyincorporated into peptides, either through the application of peptidesynthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention willgenerally be on the order of about 8 to about 20 amino acids in length,and more preferably about 8 to about 15 amino acids in length. It isproposed that shorter antigenic crystal protein-derived peptides willprovide advantages in certain circumstances, for example, in thepreparation of immunologic detection assays. Exemplary advantagesinclude the ease of preparation and purification, the relatively lowcost and improved reproducibility of production, and advantageousbiodistribution.

It is proposed that particular advantages of the present invention maybe realized through the preparation of synthetic peptides which includemodified and/or extended epitopic/immunogenic core sequences whichresult in a “universal” epitopic peptide directed to crystal proteins,and in particular Cry and Cry-related sequences. These epitopic coresequences are identified herein in particular aspects as hydrophilicregions of the particular polypeptide antigen. It is proposed that theseregions represent those which are most likely to promote T-cell orB-cell stimulation, and, hence, elicit specific antibody production.

An epitopic core sequence, as used herein, is a relatively short stretchof amino acids that is “complementary” to, and therefore will bind,antigen binding sites on the crystal protein-directed antibodiesdisclosed herein. Additionally or alternatively, an epitopic coresequence is one that will elicit antibodies that are cross-reactive withantibodies directed against the peptide compositions of the presentinvention. It will be understood that in the context of the presentdisclosure, the term “complementary” refers to amino acids or peptidesthat exhibit an attractive force towards each other. Thus, certainepitope core sequences of the present invention may be operationallydefined in terms of their ability to compete with or perhaps displacethe binding of the desired protein antigen with the correspondingprotein-directed antisera.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence anticipated by the present disclosure would generally be on theorder of about 8 amino acids in length, with sequences on the order of10 to 20 being more preferred. Thus, this size will generally correspondto the smallest peptide antigens prepared in accordance with theinvention. However, the size of the antigen may be larger where desired,so long as it contains a basic epitopic core sequence.

The identification of epitopic core sequences is known to those of skillin the art, for example, as described in U.S. Pat. No. 4,554,101,incorporated herein by reference, which teaches the identification andpreparation of epitopes from amino acid sequences on the basis ofhydrophilicity. Moreover, numerous computer programs are available foruse in predicting antigenic portions of proteins (see e.g., Jameson andWolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysisprograms (e.g., DNAStar® software, DNAStar, Inc., Madison, Wis.) mayalso be useful in designing synthetic peptides in accordance with thepresent disclosure.

Syntheses of epitopic sequences, or peptides which include an antigenicepitope within their sequence, are readily achieved using conventionalsynthetic techniques such as the solid phase method (e.g., through theuse of commercially available peptide synthesizer such as an AppliedBiosystems Model 430A Peptide Synthesizer). Peptide antigens synthesizedin this manner may then be aliquotted in predetermined amounts andstored in conventional manners, such as in aqueous solutions or, evenmore preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of about 7.0 to about 7.5.Moreover, it may be desirable to include agents which will inhibitmicrobial growth, such as sodium azide or Merthiolate. For extendedstorage in an aqueous state it will be desirable to store the solutionsat about 4° C., or more preferably, frozen. Of course, where thepeptides are stored in a lyophilized or powdered state, they may bestored virtually indefinitely, e.g., in metered aliquots that may berehydrated with a predetermined amount of water (preferably distilled)or buffer prior to use.

2.11 Crystal Protein Compositions As Insecticides and Methods of Use

The inventors contemplate that the crystal protein compositionsdisclosed herein will find particular utility as insecticides fortopical and/or systemic application to field crops, grasses, fruits andvegetables, and ornamental plants. In a preferred embodiment, thebioinsecticide composition comprises an oil flowable suspension ofbacterial cells which expresses a novel crystal protein disclosedherein. Preferably the cells are B. thuringiensis EG4096, EG 11494, orEG11502 cells, however, any such bacterial host cell expressing thenovel nucleic acid segments disclosed herein and producing a crystalprotein is contemplated to be useful, such as B. megaterium, B.subtilis, E. coli, or Pseudomonas spp.

In another important embodiment, the bioinsecticide compositioncomprises a water dispersible granule. This granule comprises bacterialcells which expresses a novel crystal protein disclosed herein.Preferred bacterial cells are B. thuringiensis EG4096, EG 11494, orEG11502 cells, however, bacteria such as B. megaterium, B. subtilis, E.coli, or Pseudomonas spp. cells transformed with a DNA segment disclosedherein and expressing the crystal protein are also contemplated to beuseful.

In a third important embodiment, the bioinsecticide compositioncomprises a wettable powder, dust, pellet, or collodial concentrate.This powder comprises bacterial cells which expresses a novel crystalprotein disclosed herein. Preferred bacterial cells are B. thuringiensisEG4096, EG11494, or EG11502 cells, however, bacteria such as B.megaterium, B. subtilis, E. coli, or Pseudomonas spp. cells transformedwith a DNA segment disclosed herein and expressing the crystal proteinare also contemplated to be useful. Such dry forms of the insecticidalcompositions may be formulated to dissolve immediately upon wetting, oralternatively, dissolve in a controlled-release, sustained release, orother time-dependent manner.

In a fourth important embodiment, the bioinsecticide compositioncomprises an aqueous suspension of bacterial cells such as thosedescribed above which express the crystal protein. Such aqueoussuspensions may be provided as a concentrated stock solution which isdiluted prior to application, or alternatively, as a diluted solutionready-to-apply.

For these methods involving application of bacterial cells, the cellularhost containing the crystal protein gene(s) may be grown in anyconvenient nutrient medium, where the DNA construct provides a selectiveadvantage, providing for a selective medium so that substantially all orall of the cells retain the B. thuringiensis gene. These cells may thenbe harvested in accordance with conventional ways. Alternatively, thecells can be treated prior to harvesting.

When the insecticidal compositions comprise intact B. thuringiensiscells expressing the protein of interest, such bacteria may beformulated in a variety of ways. They may be employed as wettablepowders, granules or dusts, by mixing with various inert materials, suchas inorganic minerals (phyllosilicates, carbonates, sulfates,phosphates, and the like) or botanical materials (powdered corncobs,rice hulls, walnut shells, and the like). The formulations may includespreader-sticker adjuvants, stabilizing agents, other pesticidaladditives, or surfactants. Liquid formulations may be aqueous-based ornon-aqueous and employed as foams, suspensions, emulsifiableconcentrates, or the like. The ingredients may include rheologicalagents, surfactants, emulsifiers, dispersants, or polymers.

Alternatively, the novel CryET29 or CryET29-derived protein may beprepared by native or recombinant bacterial expression systems in vitroand isolated for subsequent field application. Such protein may beeither in crude cell lysates, suspensions, colloids, etc., oralternatively may be purified, refined, buffered, and/or furtherprocessed, before formulating in an active biocidal formulation.Likewise, under certain circumstances, it may be desirable to isolatecrystals and/or spores from bacterial cultures expressing the crystalprotein and apply solutions, suspensions, or collodial preparations ofsuch crystals and/or spores as the active bioinsecticidal composition.

Regardless of the method of application, the amount of the activecomponent(s) are applied at an insecticidally-effective amount, whichwill vary depending on such factors as, for example, the specificcoleopteran insects to be controlled, the specific plant or crop to betreated, the environmental conditions, and the method, rate, andquantity of application of the insecticidally-active composition.

The insecticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, dessicated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,e.g., inert components, dispersants, surfactants, tackifiers, binders,etc. that are ordinarily used in insecticide formulation technology;these are well known to those skilled in insecticide formulation. Theformulations may be mixed with one or more solid or liquid adjuvants andprepared by various means, e.g., by homogeneously mixing, blendingand/or grinding the insecticidal composition with suitable adjuvantsusing conventional formulation techniques.

The insecticidal compositions of this invention are applied to theenvironment of the target coleopteran insect, typically onto the foliageof the plant or crop to be protected, by conventional methods,preferably by spraying. The strength and duration of insecticidalapplication will be set with regard to conditions specific to theparticular pest(s), crop(s) to be treated and particular environmentalconditions. The proportional ratio of active ingredient to carrier willnaturally depend on the chemical nature, solubility, and stability ofthe insecticidal composition, as well as the particular formulationcontemplated.

Other application techniques, e.g., dusting, sprinkling, soaking, soilinjection, seed coating, seedling coating, spraying, aerating, misting,atomizing, and the like, are also feasible and may be required undercertain circumstances such as e.g., insects that cause root or stalkinfestation, or for application to delicate vegetation or ornamentalplants. These application procedures are also well-known to those ofskill in the art.

The insecticidal composition of the invention may be employed in themethod of the invention singly or in combination with other compounds,including and not limited to other pesticides. The method of theinvention may also be used in conjunction with other treatments such assurfactants, detergents, polymers or time-release formulations. Theinsecticidal compositions of the present invention may be formulated foreither systemic or topical use.

The concentration of insecticidal composition which is used forenvironmental, systemic, or foliar application will vary widelydepending upon the nature of the particular formulation, means ofapplication, environmental conditions, and degree of biocidal activity.Typically, the bioinsecticidal composition will be present in theapplied formulation at a concentration of at least about 1% by weightand may be up to and including about 99% by weight. Dry formulations ofthe compositions may be from about 1% to about 99% or more by weight ofthe composition, while liquid formulations may generally comprise fromabout 1% to about 99% or more of the active ingredient by weight.Formulations which comprise intact bacterial cells will generallycontain from about 10⁴ to about 10¹² cells/mg.

The insecticidal formulation may be administered to a particular plantor target area in one or more applications as needed, with a typicalfield application rate per hectare ranging on the order of from about 50g to about 500 g of active ingredient, or of from about 500 g to about1000 g, or of from about 1000 g to about 5000 g or more of activeingredient.

2.12 Pharmaceutical Compositions and Methods for the Treatment of Fleas

Since the novel crystal protein of the present invention is the firstsuch B. thuringiensis δ-endotoxin identified which has insecticidalactivity against fleas, the inventors also contemplate the formulationof pharmaceutical compositions which may be given to animals asprophylaxis and/or treatment of infestation by fleas, and in particularby infestation of members of the Genus Ctenocephalides, such asCtenocephalides felis (common name, cat flea) and C. canis (common name,dog flea). While these are only two members of the Order Siphonapterafor which the present invention's compositions demonstrate insecticidalactivity, it is contemplated that the compositions may be useful intreating other related insects which commonly attack animals may also becontrolled by the novel compositions disclosed herein. Such insects aredescribed in detail in U.S. Pat. No. 5,449,681, incorporated herein byreference, and include members of the Genera Culex, Culiseta, Bovicola,Callitroga, Chrysops, Cimes, Ctenocephalis, Dermatophilus, Dermatobia,and Damalinia among others.

As such, one aspect of the invention comprises a pharmaceuticalcomposition comprising a crystal protein composition disclosed hereinfor administration to an animal to prevent or reduce flea or relatedinsect infestation. A method of reducing such flea infestation in ananimal is also disclosed and claimed herein. The method generallycomprises administering to an animal an insecticidally-effective amountof a CryET29 composition. Means for administering such insecticidalcompositions to an animal are well-known in the art. U.S. Pat. No.5,416,102 (specifically incorporated herein by reference) providesteaching for methods and formulations for preventing flea infestationusing an insecticidal composition.

Such anti-siphonapteran veterinary compositions may be delivered in avariety of methods depending upon the particular application. Examplesof means for administering insecticidal compositions to an animal arewell-known to those of skill in the art, and include, e.g., fleacollars, flea sprays, dips, powders and the like. Methods for providingsuch formulations to an animal are also well-known to those of skill inthe art, and include direct application or passive application such asthe device described in U.S. Pat. No. 4,008,688 for the application ofinsecticides by a pet bed assembly. The animal to be treated may be anyanimal which is sensitive to or susceptible to attack or infestation bya flea which is killed or inhibited by a CryET29 composition asdisclosed herein. Such animals may be feline, canine, equine, porcine,lupine, bovine, murine, etc. and the like, although the inventorscontemplate that feline and canine animals will be particularlypreferred as animals to be treated by the novel compositions disclosedherein.

It is further contemplated that in addition to topical administration ofthe pharmaceutical compositions disclosed, systemic administration mayin some cases be preferable or desirable. For oral administration, thecompositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet. For oraltherapeutic administration, the active compounds may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltables, troches, capsules, elixirs, suspensions, syrups, wafers, and thelike. Such compositions and preparations should contain at least 0.1% ofactive compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 2 toabout 60% of the weight of the unit. The amount of active compounds insuch therapeutically useful compositions is such that a suitable dosagewill be obtained.

For oral prophylaxis of fleas, the crystal protein may be incorporatedwith excipients and used in the form of a gel, paste, powder, pill,tablet, capsule, or slurry which may be given to the animal foringestion. Alternatively the compositions may be formulated as anadditive to pet foods, treats, or other edible formulations. Whenformulated as a tablet or capsule, or the like, the composition may alsocontain the following: a binder, as gum tragacanth, acacia, cornstarch,or gelatin; excipients, such as dicalcium phosphate; a disintegratingagent, such as corn starch, potato starch, alginic acid and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin may be added or a flavoring agent to makethe composition more palatable to the animal being treated. One suchmeans for delivering flea prophylactics to an animal is a sauce asdescribed in U.S. Pat. No. 4,702,914, specifically incorporated hereinby reference.

When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. Of course, any material used in preparingany dosage unit form should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the active compounds maybe incorporated into sustained-release preparation and formulations.

Alternatively, the pharmaceutical compositions disclosed herein may beadministered parenterally, intramuscularly, or even intraperitoneally.Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), suitable mixtures thereof, and/orvegetable oils. Proper fluidity may be maintained, for example, by theuse of a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.The prevention of the action of microorganisms can be brought about byvarious antibacterial ad antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

When systemic administration is desired, e.g., parenteral administrationin an aqueous solution, the solution should be suitably buffered ifnecessary and the liquid diluent first rendered isotonic with sufficientsaline or glucose. These particular aqueous solutions are especiallysuitable for intramuscular, subcutaneous and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. Some variation in dosage will necessarily occurdepending on the condition, size, and type of animal being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas creams, lotions, sprays, dips, emulsions, colloids, or alternatively,when systemic administration is desirable, injectable solutions, drugrelease capsules and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a animal. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

Another aspect of the invention encompasses methods and compositions foruse in the control and eradication of siphonapteran insects fromenvironmental areas where infestation by such insects is suspected. Themethod generally involves applying to an area suspected of containingsuch insects an insecticidally-effective amount of a CryET29 compositionas disclosed herein. The inventors further contemplate the use of theprotein of the present invention as an active ingredient in apharmaceutical composition for administration to body or to the livingareas and environs of an animal to prevent, lessen, or reduce theinfestation of fleas and related insects in such areas. The crystalprotein composition may be formulated in a powder, spray, fog, granule,rinse, shampoo, flea collar, dip, etc. suitable for administration tothe body of the animal or to the living quarters, bedding materials,houses, yards, kennels, pet boarding facilities etc. of such an animalusing techniques which are known to those of skill in the art ofveterinary insecticide formulations. An example of oral formulation ofveterinary insecticides is found in the teachings of U.S. Pat. No.5,416,102. The inventors contemplate that the use of such compositionsin the prevention or eradication of fleas on pets such as dogs, cats,and other fur-bearing animals may represent a significant advance in thestate of the art considering the novel compositions disclosed herein arethe first crystal proteins identified which have such desirableanti-siphonapteran insecticidal activity.

2.13 Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. In particular embodiments ofthe invention, mutated crystal proteins are contemplated to be usefulfor increasing the insecticidal activity of the protein, andconsequently increasing the insecticidal activity and/or expression ofthe recombinant transgene in a plant cell. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thecodons given in Table 2. TABLE 2 Amino Acids Codons Alanine Ala A GCAGCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Giutamicacid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGGGGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys KAAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

3. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A and FIG. 1B show the nucleic acid sequence of the cryET29 gene(SEQ ID NO:1), and the corresponding deduced amino acid sequence of theCryET29 protein (SEQ ID NO:2).

4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides a novel δ-endotoxin, designated CryET29,which is toxic to larvae of the cat flea, Ctenocephalides felis, as wellas against coleopteran insects such as the southern and western cornrootworm, Colorado potato beetle, Japanese beetle, and the red flourbeetle. It is important to note that the trivial name forCtenocephalides felis is somewhat misleading in that the organismparasitizes not only felines, but is the major parasitic flea forcanines as well (see e.g., U.S. Pat. No. 4,547,360, specificallyincorporated herein by reference).

4.1 cryET29 DNA Probes and Primers

In another aspect, DNA sequence information provided by the inventionallows for the preparation of relatively short DNA (or RNA) sequenceshaving the ability to specifically hybridize to gene sequences of theselected polynucleotides disclosed herein. In these aspects, nucleicacid probes of an appropriate length are prepared based on aconsideration of a selected crystal protein gene sequence, e.g., asequence such as that shown in SEQ ID NO: 1. The ability of such nucleicacid probes to specifically hybridize to a crystal protein-encoding genesequence lends them particular utility in a variety of embodiments. Mostimportantly, the probes may be used in a variety of assays for detectingthe presence of complementary sequences in a given sample.

In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying or mutating adefined segment of a crystal protein gene from B. thuringiensis usingPCR™ technology. Segments of related crystal protein genes from otherspecies may also be amplified by PCR™ using such primers.

4.2 Expression Vectors

The present invention contemplates an expression vector comprising apolynucleotide of the present invention. Thus, in one embodiment anexpression vector is an isolated and purified DNA molecule comprising apromoter operatively linked to an coding region that encodes apolypeptide of the present invention, which coding region is operativelylinked to a transcription-terminating region, whereby the promoterdrives the transcription of the coding region.

As used herein, the term “operatively linked” means that a promoter isconnected to an coding region in such a way that the transcription ofthat coding region is controlled and regulated by that promoter. Meansfor operatively linking a promoter to a coding region are well known inthe art.

In a preferred embodiment, the recombinant expression of DNAs encodingthe crystal proteins of the present invention is preferable in aBacillus host cell. Preferred host cells include B. thuringiensis, B.megaterium, B. subtilis, and related bacilli, with B. thuringiensis hostcells being highly preferred. Promoters that function in bacteria arewell-known in the art. An exemplary and preferred promoter for theBacillus crystal proteins include any of the known crystal protein genepromoters, including the cryET29 gene promoter, and promoters specificfor B. thuringiensis sigma factors, such as σ^(E) and σ^(K) (for areview see Baum and Malvar, 1995) Alternatively, mutagenized orrecombinant crystal protein-encoding gene promoters may be engineered bythe hand of man and used to promote expression of the novel genesegments disclosed herein.

In an alternate embodiment, the recombinant expression of DNAs encodingthe crystal proteins of the present invention is performed using atransformed Gram-negative bacterium such as an E. coli or Pseudomonasspp. host cell. Promoters which function in high-level expression oftarget polypeptides in E. coli and other Gram-negative host cells arealso well-known in the art.

Where an expression vector of the present invention is to be used totransform a plant, a promoter is selected that has the ability to driveexpression in plants. Promoters that function in plants are also wellknown in the art. Useful in expressing the polypeptide in plants arepromoters that are inducible, viral, synthetic, constitutive asdescribed (Poszkowski et al., 1989; Odell et al., 1985), and temporallyregulated, spatially regulated, and spatio-temporally regulated (Chau etal., 1989).

A promoter is also selected for its ability to direct the transformedplant cell's or transgenic plant's transcriptional activity to thecoding region. Structural genes can be driven by a variety of promotersin plant tissues. Promoters can be near-constitutive, such as the CaMV35S promoter, or tissue-specific or developmentally specific promotersaffecting dicots or monocots.

Where the promoter is a near-constitutive promoter such as CaMV 35S,increases in polypeptide expression are found in a variety oftransformed plant tissues (e.g., callus, leaf, seed and root).Alternatively, the effects of transformation can be directed to specificplant tissues by using plant integrating vectors containing atissue-specific promoter.

An exemplary tissue-specific promoter is the lectin promoter, which isspecific for seed tissue. The Lectin protein in soybean seeds is encodedby a single gene (Le1) that is only expressed during seed maturation andaccounts for about 2 to about 5% of total seed mRNA. The lectin gene andseed-specific promoter have been fully characterized and used to directseed specific expression in transgenic tobacco plants (Vodkin et al.,1983; Lindstrom et al., 1990.)

An expression vector containing a coding region that encodes apolypeptide of interest is engineered to be under control of the lectinpromoter and that vector is introduced into plants using, for example, aprotoplast transformation method (Dhir et al., 1991). The expression ofthe polypeptide is directed specifically to the seeds of the transgenicplant.

A transgenic plant of the present invention produced from a plant celltransformed with a tissue specific promoter can be crossed with a secondtransgenic plant developed from a plant cell transformed with adifferent tissue specific promoter to produce a hybrid transgenic plantthat shows the effects of transformation in more than one specifictissue.

Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yanget al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989), cornlight harvesting complex (Simpson, 1986), corn heat shock protein (Odellet al., 1985), pea small subunit RuBP Carboxylase (Poulsen et al., 1986;Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al.,1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petuniachalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1(Keller et al., 1989), CaMV 35s transcript (Odell et al., 1985) andPotato patatin (Wenzler et al., 1989). Preferred promoters are thecauliflower mosaic virus (CaMV 35S) promoter and the S-E9 small subunitRuBP carboxylase promoter.

The choice of which expression vector and ultimately to which promoter apolypeptide coding region is operatively linked depends directly on thefunctional properties desired, e.g., the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the expression of the polypeptide coding regionto which it is operatively linked.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of Agrobacterium tumefaciens described (Rogers et al.,1987). However, several other plant integrating vector systems are knownto function in plants including pCaMVCN transfer control vectordescribed (Fromm et al., 1985). Plasmid pCaMVCN (available fromPharmacia, Piscataway, N.J.) includes the cauliflower mosaic virus CaMV35S promoter.

In preferred embodiments, the vector used to express the polypeptideincludes a selection marker that is effective in a plant cell,preferably a drug resistance selection marker. One preferred drugresistance marker is the gene whose expression results in kanamycinresistance; i.e., the chimeric gene containing the nopaline synthasepromoter, Tn5 neomycin phosphotransferase II (nptII) and nopalinesynthase 3′ nontranslated region described (Rogers et al., 1988).

RNA polymerase transcribes a coding DNA sequence through a site wherepolyadenylation occurs. Typically, DNA sequences located a few hundredbase pairs downstream of the polyadenylation site serve to terminatetranscription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of transcribed messenger RNA (mRNA).

Means for preparing expression vectors are well known in the art.Expression (transformation vectors) used to transform plants and methodsof making those vectors are described in U.S. Patent Nos. 4,971,908,4,940,835, 4,769,061 and 4,757,011, the disclosures of which areincorporated herein by reference. Those vectors can be modified toinclude a coding sequence in accordance with the present invention.

A variety of methods has been developed to operatively link DNA tovectors via complementary cohesive termini or blunt ends. For instance,complementary homopolymer tracts can be added to the DNA segment to beinserted and to the vector DNA. The vector and DNA segment are thenjoined by hydrogen bonding between the complementary homopolymeric tailsto form recombinant DNA molecules.

A coding region that encodes a polypeptide having the ability to conferinsecticidal activity to a cell is preferably a CryET29 B. thuringiensiscrystal protein-encoding gene. In preferred embodiments, such apolypeptide has the amino acid residue sequence of SEQ ID NO:2, or afunctional equivalent of this sequence. In accordance with suchembodiments, a coding region comprising the DNA sequence of SEQ ID NO: 1is also preferred.

4.3 Characteristics of the CryET29 Crystal Protein

The present invention provides novel polypeptides that define a whole ora portion of a B. thuringiensis CryET29 crystal protein.

In a preferred embodiment, the invention discloses and claims anisolated and purified CryET29 protein. The CryET29 protein comprises anamino acid sequence as disclosed in SEQ ID NO:2. CryET29 has acalculated isoelectric constant (pI) equal to 5.88. The amino acidcomposition of the CryET29 protein is given in Table 3. TABLE 3 AminoAcid Composition of CryET29 Amino Acid # Residues % Total Ala 18 7.7 Arg7 3.0 Asn 15 6.4 Asp 15 6.4 Cys 1 0.4 Gln 15 6.4 Glu 10 4.3 Gly 5 2.1His 3 1.2 Ile 20 8.6 Leu 13 5.6 Lys 16 6.9 Met 4 1.7 Phe 12 5.1 Pro 62.5 Ser 16 6.9 Thr 17 7.3 Trp 2 0.8 Tyr 10 4.3 Val 26 11.2 Acidic (Asp +Glu) 25 10.7 Basic (Arg + Lys) 23 9.9 Aromatic (Phe + Trp + Tyr) 24 10.2Hydrophobic (Aromatic + Ile + Leu + Met + Val) 87 37.34.4 Transformed or Transgenic Plant Cells

A bacterium, a yeast cell, or a plant cell or a plant transformed withan expression vector of the present invention is also contemplated. Atransgenic bacterium, yeast cell, plant cell or plant derived from sucha transformed or transgenic cell is also contemplated. Means fortransforming bacteria and yeast cells are well known in the art.Typically, means of transformation are similar to those well known meansused to transform other bacteria or yeast such as E. coli orSaccharomyces cerevisiae.

Methods for DNA transformation of plant cells includeAgrobacterium-mediated plant transformation, protoplast transformation,gene transfer into pollen, injection into reproductive organs, injectioninto immature embryos and particle bombardment. Each of these methodshas distinct advantages and disadvantages. Thus, one particular methodof introducing genes into a particular plant strain may not necessarilybe the most effective for another plant strain, but it is well knownwhich methods are useful for a particular plant strain.

There are many methods for introducing transforming DNA segments intocells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as by Agrobacterium infection,direct delivery of DNA such as, for example, by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

Technology for introduction of DNA into cells is well-known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and van der Eb, 1973;Zatloukal et al., 1992); (2) physical methods such as microinjection(Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al.,1985) and the gene gun (Johnston and Tang, 1994; Fynan et al., 1993);(3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson,1988a; 1988b); and (4) receptor-mediated mechanisms (Curiel et al., 1991; 1992; Wagner et al., 1992).

4.5.1 Electroporation

The application of brief, high-voltage electric pulses to a variety ofanimal and plant cells leads to the formation of nanometer-sized poresin the plasma membrane. DNA is taken directly into the cell cytoplasmeither through these pores or as a consequence of the redistribution ofmembrane components that accompanies closure of the pores.Electroporation can be extremely efficient and can be used both fortransient expression of clones genes and for establishment of cell linesthat carry integrated copies of the gene of interest. Electroporation,in contrast to calcium phosphate-mediated transfection and protoplastfusion, frequently gives rise to cell lines that carry one, or at most afew, integrated copies of the foreign DNA.

The introduction of DNA by means of electroporation, is well-known tothose of skill in the art. In this method, certain cell wall-degradingenzymes, such as pectin-degrading enzymes, are employed to render thetarget recipient cells more susceptible to transformation byelectroporation than untreated cells. Alternatively, recipient cells aremade more susceptible to transformation, by mechanical wounding. Toeffect transformation by electroporation one may employ either friabletissues such as a suspension culture of cells, or embryogenic callus, oralternatively, one may transform immature embryos or other organizedtissues directly. One would partially degrade the cell walls of thechosen cells by exposing them to pectin-degrading enzymes (pectolyases)or mechanically wounding in a controlled manner. Such cells would thenbe recipient to DNA transfer by electroporation, which may be carriedout at this stage, and transformed cells then identified by a suitableselection or screening protocol dependent on the nature of the newlyincorporated DNA.

4.5.2 Microprojectile Bombardment

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming monocots, is thatneither the isolation of protoplasts (Cristou et al., 1988) nor thesusceptibility to Agrobacterium infection is required. An illustrativeembodiment of a method for delivering DNA into a monocot cell byacceleration is a Biolistics Particle Delivery System, which can be usedto propel particles coated with DNA or cells through a screen, such as astainless steel or Nytex screen, onto a filter surface covered with corncells cultured in suspension. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectiles aggregate andmay contribute to a higher frequency of transformation by reducingdamage inflicted on the recipient cells by projectiles that are toolarge.

For the bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth herein one may obtainup to 1000 or more foci of cells transiently expressing a marker gene.The number of cells in a focus which express the exogenous gene product48 hours post-bombardment often range from 1 to 10 and average 1 to 3.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance, and helium pressure.One may also minimize the trauma reduction factors (TRFs) by modifyingconditions which influence the physiological state of the recipientcells and which may therefore influence transformation and integrationefficiencies. For example, the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient cells may be adjustedfor optimum transformation. The execution of other routine adjustmentswill be known to those of skill in the art in light of the presentdisclosure.

4.5.3 Agrobacterium-Mediated Transfer

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al., 1987). Further, the integration of the Ti-DNAis a relatively precise process resulting in few rearrangements. Theregion of DNA to be transferred is defined by the border sequences, andintervening DNA is usually inserted into the plant genome as described(Spielmann et al., 1986; Jorgensen et al., 1987).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described (Rogers et al.,1987), have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

Agrobacterium-mediated transformation of leaf disks and other tissuessuch as cotyledons and hypocotyls appears to be limited to plants thatAgrobacterium naturally infects. Agrobacterium-mediated transformationis most efficient in dicotyledonous plants. Few monocots appear to benatural hosts for Agrobacterium, although transgenic plants have beenproduced in asparagus using Agrobacterium vectors as described (Bytebieret al., 1987). Therefore, commercially important cereal grains such asrice, corn, and wheat must usually be transformed using alternativemethods. However, as mentioned above, the transformation of asparagususing Agrobacterium can also be achieved (see, for example, Bytebier etal., 1987).

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome. Such transgenicplants can be referred to as being heterozygous for the added gene.However, inasmuch as use of the word “heterozygous” usually implies thepresence of a complementary gene at the same locus of the secondchromosome of a pair of chromosomes, and there is no such gene in aplant containing one added gene as here, it is believed that a moreaccurate name for such a plant is an independent segregant, because theadded, exogenous gene segregates independently during mitosis andmeiosis.

More preferred is a transgenic plant that is homozygous for the addedstructural gene; i.e., a transgenic plant that contains two added genes,one gene at the same locus on each chromosome of a chromosome pair. Ahomozygous transgenic plant can be obtained by sexually mating (selfing)an independent segregant transgenic plant that contains a single addedgene, germinating some of the seed produced and analyzing the resultingplants produced for enhanced insecticidal activity relative to a control(native, non-transgenic) or an independent segregant transgenic plant.

It is to be understood that two different transgenic plants can also bemated to produce offspring that contain two independently segregatingadded, exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both added, exogenous genes that encode apolypeptide of interest. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated.

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1986; Uchimiyaet al., 1986; Callis et al., 1987; Marcotte et al., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastsare described (Fujimura et al., 1985; Toriyama et al., 1986; Yamada etal., 1986; Abdullah et al., 1986).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, 1988). Inaddition, “particle gun” or high-velocity microprojectile technology canbe utilized (Vasil, 1992).

Using that latter technology, DNA is carried through the cell wall andinto the cytoplasm on the surface of small metal particles as described(Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988). The metalparticles penetrate through several layers of cells and thus allow thetransformation of cells within tissue explants.

4.6 Methods for Producing Insect-Resistant Transgenic Plants

By transforming a suitable host cell, such as a plant cell, with arecombinant cryET29 gene-containing segment, the expression of theencoded crystal protein (i.e., a bacterial crystal protein orpolypeptide having insecticidal activity against coleopterans) canresult in the formation of insect-resistant plants.

By way of example, one may utilize an expression vector containing acoding region for a B. thuringiensis crystal protein and an appropriateselectable marker to transform a suspension of embryonic plant cells,such as wheat or corn cells using a method such as particle bombardment(Maddock et al., 1991; Vasil et al., 1992) to deliver the DNA coated onmicroprojectiles into the recipient cells. Transgenic plants are thenregenerated from transformed embryonic calli that express theinsecticidal proteins.

The formation of transgenic plants may also be accomplished using othermethods of cell transformation which are known in the art such asAgrobacterium-mediated DNA transfer (Fraley et al., 1983).Alternatively, DNA can be introduced into plants by direct DNA transferinto pollen (Zhou et al., 1983; Hess, 1987; Luo et al., 1988), byinjection of the DNA into reproductive organs of a plant (Pena et al.,1987), or by direct injection of DNA into the cells of immature embryosfollowed by the rehydration of desiccated embryos (Neuhaus et al., 1987;Benbrook et al., 1986).

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach and Weissbach, 1988). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983).

This procedure typically produces shoots within two to four months andthose shoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Shoots that rooted in the presence of the selective agent toform plantlets are then transplanted to soil or other media to allow theproduction of roots. These procedures vary depending upon the particularplant strain employed, such variations being well known in the art.Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, as discussed before. Otherwise, pollenobtained from the regenerated plants is crossed to seed-grown plants ofagronomically important, preferably inbred lines. Conversely, pollenfrom plants of those important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

A transgenic plant of this invention thus has an increased amount of acoding region (e.g., a cry gene) that encodes the Cry polypeptide ofinterest. A preferred transgenic plant is an independent segregant andcan transmit that gene and its activity to its progeny. A more preferredtransgenic plant is homozygous for that gene, and transmits that gene toall of its offspring on sexual mating. Seed from a transgenic plant maybe grown in the field or greenhouse, and resulting sexually maturetransgenic plants are self-pollinated to generate true breeding plants.The progeny from these plants become true breeding lines that areevaluated for, by way of example, increased insecticidal capacityagainst coleopteran insects and cat flea larvae, preferably in thefield, under a range of environmental conditions. The inventorscontemplate that the present invention will find particular utility inthe creation of transgenic plants of commercial interest includingvarious turf grasses, wheat, corn, rice, barley, oats, a variety ofornamental plants and vegetables, as well as a number of nut- andfruit-bearing trees and plants.

4.7 Nomenclature of the Novel Proteins

The inventors have arbitrarily assigned the designation CryET29 to thenovel protein of the invention. Likewise, the arbitrary designation ofcryET29 has been assigned to the novel nucleic acid sequence whichencodes this polypeptide. Formal assignment of the gene and proteindesignations based on the revised nomenclature of crystal proteinendotoxins (Table 1) will be assigned by a committee on the nomenclatureof B. thuringiensis, formed to systematically classify B. thuringiensiscrystal proteins. The inventors contemplate that the arbitrarilyassigned designations of the present invention will be superseded by theofficial nomenclature assigned to these sequences.

4.8 Definitions

The following words and phrases have the meanings set forth below.

Expression

The combination of intracellular processes, including transcription andtranslation undergone by a coding DNA molecule such as a structural geneto produce a polypeptide.

Promoter

A recognition site on a DNA sequence or group of DNA sequences thatprovide an expression control element for a structural gene and to whichRNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

Regeneration

The process of growing a plant from a plant cell (e.g., plant protoplastor explant).

Structural gene

A gene that is expressed to produce a polypeptide.

Transformation

A process of introducing an exogenous DNA sequence (e.g., a vector, arecombinant DNA molecule) into a cell or protoplast in which thatexogenous DNA is incorporated into a chromosome or is capable ofautonomous replication.

Transformed cell

A cell whose DNA has been altered by the introduction of an exogenousDNA molecule into that cell.

Transgenic cell

Any cell derived or regenerated from a transformed cell or derived froma transgenic cell. Exemplary transgenic cells include plant calliderived from a transformed plant cell and particular cells such as leaf,root, stem, e.g., somatic cells, or reproductive (germ) cells obtainedfrom a transgenic plant.

Transgenic plant

A plant or progeny thereof derived from a transformed plant cell orprotoplast, wherein the plant DNA contains an introduced exogenous DNAmolecule not originally present in a native, non-transgenic plant of thesame strain. The terms “transgenic plant”and “transformed plant” havesometimes been used in the art as synonymous terms to define a plantwhose DNA contains an exogenous DNA molecule. However, it is thoughtmore scientifically correct to refer to a regenerated plant or callusobtained from a transformed plant cell or protoplast as being atransgenic plant, and that usage will be followed herein.

Vector

A DNA molecule capable of replication in a host cell and/or to whichanother DNA segment can be operatively linked so as to bring aboutreplication of the attached segment. A plasmid is an exemplary vector.

5. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

5.1 Example 1 Isolation of B. thuringiensis EG4096

Crop dust samples were obtained from various sources throughout the U.S.and abroad, typically grain storage facilities. The crop dust sampleswere treated and spread on agar plates to isolate individualBacillus-type colonies as described (Donovan et al., 1993). EG4096 is awild-type B. thuringiensis strain isolated from a crop dust sample fromThailand. Phase contrast microscopy was used to visually examine thecrystal morphology of the bacterial colonies from this crop dust. Thecolony designated EG4096 contained endospores and crystalline inclusionsof a unique morphology resembling short needles. The array of plasmidsnative to strain EG4096 is also unique.

Insect bioassay of this wild-type B. thuringiensis strain determinedthat it had insecticidal activity against larvae of coleopteran insects,including Southern corn rootworm, western corn rootworm, Colorado potatobeetle, red flour beetle, and Japanese beetle. EG4096 also exhibitsinsecticidal activity against larva of the cat flea.

Characterization of EG4096 included the analysis of crystal proteinproduced by the strain during sporulation and the cloning and expressionof the gene encoding the crystal protein, which has been designatedcryET29. The insecticidal activity of both the wild-type strain and of arecombinant B. thuringiensis expressing the cloned cryET29 toxin genewas determined.

5.2 Example 2 Evaluation of the Native Plasmids of B. thuringiensisStrain EG4096

The complement of native plasmids contained within isolated B.thuringiensis EG4096 was determined by modified Eckhardt agarose gelelectrophoresis as described by Gonzalez et al., (1982). The pattern ofnative plasmids did not correspond to patterns of typical known serovars(Carlton and Gonzalez, 1985). The plasmid sizes are 5.0, 7.2, 6.0 (opencircular), 39, 80 and 100 MDa.

5.3 Example 3 Evaluation of the Crystal Protein of B. thuringiensisEG4096

EG4096 was grown in DSM+ glucose sporulation medium [0.8% (wt/vol) Difconutrient broth, 0.5% (wt/vol) glucose, 10 mM K₂HPO₄, 10 mM KH₂PO₄, 1 mMCa(NO₃)₂, 0.5 mM MgSO₄, 10 μM MnCl₂, 10 μM FeSO₄] for three days at 30°C. during which the culture grew to stationary phase, sporulated andlysed, thus releasing the protein inclusions into the medium. Thecultures were harvested by centrifugation which pelleted the spores andcrystals. The pellet was washed in a solution of 0.005% Triton X-100®, 2mM EDTA and recentrifuged. The washed pellet was resuspended atone-tenth the original volume of 0.005% Triton X-100®, 2 mM EDTA.

Crystal protein was solubilized from the spores-crystals suspension byincubating the suspension in solubilization buffer [0.14 M Tris-HCl pH8.0, 2% (wt/vol) sodium dodecyl sulfate (SDS), 5% (vol/vol)2-mercaptoethanol, 10% (vol/vol) glycerol, and 0.1% bromphenol blue] at100° C. for 5 min. The solubilized crystal protein was size fractionatedby SDS-PAGE. After size fractionation the proteins were visualized byCoomassie Brilliant Blue R-250 staining. This analysis showed that themajor crystal protein present in sporulated cultures of EG4096 isapproximately 25-kDa in size. This novel protein was designated CryET29.

To further characterize CryET29, the NH₂-terminal amino acid sequence ofthe protein was determined. A sporulated culture of EG4096 was washedand resuspended. The suspension was solubilized and run on an acrylamidegel following the procedures for SDS-PAGE analysis. Afterelectrophoresis the proteins were transferred to a BioRad PVDF membranefollowing standard western blotting procedures. After transfer, themembrane was rinsed 3− in dH₂O and washed in Amido Black 1013 stain for1 min (Sigma Chemical Co., St. Louis, Mo.). The filter was destained 1min in 5% acetic acid and then rinsed in 3 changes of dH₂O. The portionof the filter containing the approximately 25-kDa protein band wasexcised with a razor blade. This procedure resulted in a pure form ofCryET29 being obtained as a protein blotted onto a PVDF membrane(BioRad, Hercules, Calif.).

The determination of the NH₂-terminal amino acid sequence of thepurified CryET29 protein immobilized on the membrane was performed inthe Department of Physiology at the Tufts Medical School, Boston, Mass.using standard automated Edman degradation procedures The NH₂-terminalsequence was determined to be: (SEQ ID NO:3)1  2  3  4  5  6  7  8  9  10 11 12 13 14 15MetPhePheAsnArgValIleThrLeuThrValProSerSerAsp

Computer algorithms (Korn and Queen, 1984) were used to compare theN-terminal sequence of the CryET29 protein with amino acid sequences ofall B. thuringiensis crystal proteins of which the inventors are awareincluding the sequences of all B. thuringiensis crystal proteins whichhave been published in scientific literature, international patentapplications, or issued patents. A list of the crystal proteins whosesequences have been published along with the source of publication isshown in Table 4. TABLE 4 B. thuringiensis Crystal Proteins Described inthe Literature Crystal Protein Source or Reference Cry1A(a) J. Biol.Chem., 260: 6264-6272 Cry1A(b) DNA, 5: 305-314 Cry1A(c) Gene, 36:289-300 Cry1B Nucl. Acids Res., 16: 4168-4169 Cry1C Nucl. Acids Res.,16: 6240 Cry1Cb Appl. Environ. Micro., 59: 1131-1137 Cry1C(b) Nucl.Acids Res., 18: 7443 Cry1D Nucl. Acids Res., 18: 5545 Cry1E EPO 358 557A2 Cry1F J. Bacteriol., 173: 3966-3976 Cry1G FEBS, 293: 25-28 CryV WO90/13651 Cry2A J. Biol. Chem., 263: 561-567 Cry2B J. Bacteriol., 171:965-974 Cry2C FEMS Microbiol. Lett., 81: 31-36 Cry3A Proc. Natl. Acad.Sci. USA, 84: 7036-7040 Cry3B Nucl. Acids Res., 18: 1305 Cry3B2 Appl.Environ. Microbiol., 58: 3921-3927 Cry3B3 U.S. Pat. No.5,378,625 Cry3CAppl. Environ. Microbiol., 58: 2536-2542 Cry3D Gene, 110: 131-132 Cry4ANucl. Acids Res., 15: 7195 Cry4B EPO 308,199 Cry4C J. Bacteriol., 166:801-811 Cry4D J. Bacteriol., 170: 4732, 1988 Cry5 Molec. Micro., 6:1211-1217 Cry33AkD WO 94/13785 Cry33BkD WO 94/13785 Cry34kD J.Bacteriol., 174: 549-557 Cry40kD J. Bacteriol., 174: 549-557 Cry201T635WO 95/02693 Cry517 J. Gen. Micro., 138: 55-62 Crya7A021 EPO 256,553 B1CryAB78ORF1 WO 94/21795 CryAB780RF2 WO 94/21795 CryAB78100kD WO 94/21795Crybtpgs1208 EPO 382 990 Crybtpgs 1245 EPO 382 990 Crybts02618A WO94/05771 CryBuibui WO 93/03154 CryET4 U.S. Pat. No.5,322,687 CryET5 U.S.Pat. No.5,322,687 CryGei87 EPO 238,441 CryHD511 U.S. Pat. No.5,286,486CryHD867 U.S. Pat. No.6,286,486 CryIPL U.S. Pat. No.5,231,008 CryMITS JP6000084 CryPS17A WO 92/19739 CryPS17B U.S. Pat. No.5,350,576 and5,424,410 CryP16 WO 95/00639 CryP18 WO 95/00639 CryP66 WO 95/00639CryPS33F2 WO 92/19739 and U.S. Pat. No.5,424,410 CryPS40D1 U.S. Pat.No.5,273,746 CryPS43F WO 93/04587 CryPS 50Ca WO 93/04587 and EPO 498,537A2 CryPS 50Cb WO 93/15206 Cryps52A1 U.S. Pat. No.4,849,217 CryPS63B WO92/19739 CryPS69D1 U.S. Pat. No.5,424,410 Cryps71M3 WO 95/02694CryPS80JJ1 WO 94/16079 CryPS81IA U.S. Pat. No.5,273,746 CryPS81IA2 EPO405 810 Cryps81A2 EPO 401 979 CryPS81IB WO 93/14641 CryPS81IB2 U.S. Pat.No.5,273,746 Cryps81f U.S. Pat. No.5,045,469 Cryps81gg U.S. Pat.No.5,273,746 Cryps81rr1 EPO 401 979 Cryps86A1 U.S. Pat. No.5,468,636CryX FEBS Lett., 336: 79-82 CryXenA24 WO 95/00647 CrycytA Nucl. AcidsRes., 13: 8207-8217

The N-terminal sequence of the CryET29 protein was not found to behomologous to any of the known B. thuringiensis crystal proteinsidentified in Table 4.

5.4 Example 4 Isolation of a DNA Fragment Comprising the B.thuringiensis EG4096 cryET29 Gene

In order to identify the gene encoding the CryET29 protein, anoligonucleotide probe specific for the NH₂-terminal amino acid sequenceof the protein was designed. Using codons typically found in B.thuringiensis toxin genes, an oligo of 35 nucleotides was synthesized byIntegrated DNA Technologies, Inc. (Coralville, IA.) and designatedwd270. The sequence of wd270 is: (SEQ ID NO:4)5′-ATGTTTTTTAATAGAGTAATTACATTAACAGTACC-3′

Radioactively-labeled wd270 was used as a probe in Southern blot studiesas described below to identify a DNA restriction fragment containing thecryET29 gene. Total DNA was extracted from EG4096 by the followingprocedure. Vegetative cells were resuspended in a lysis buffercontaining 50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 4mg/ml lysozyme. The suspension was incubated at 37° C. for one hr.Following incubation, the suspension was extracted with an equal volumeof phenol, one time with an equal volume of phenol:chloroform:isoamylalcohol (50:48:2), and once with an equal volume of chloroform:isoamylalcohol (24:1). DNA was precipitated from the aqueous phase by theaddition of one-tenth volume 3 M sodium acetate then two volumes of 100%ethanol. The precipitated DNA was collected by centrifugation, washedwith 70% ethanol and resuspended in dH₂O.

The extracted DNA was then digested, in separate reactions, with variousrestriction endonucleases, including EcoRI. The digested DNA was sizefractionated by electrophoresis through an 0.8% agarose gel in 1X TBEovernight at 2 V/cm. The fractionated DNA fragments were transferred toan Immobilon-NC nitrocellulose filter (Millipore Corp., Bedford, Mass.)according to the method of Southern (1975). DNA was fixed to the filterby baking at 80° C. in a vacuum oven.

To identify the DNA fragment(s) containing the sequence encoding theNH₂-terminus of the CryET29 protein (see Example 3) the oligonucleotidewd270 was radioactively labeled at the 5′ ends and used as ahybridization probe. To radioactively label the probe, 1 to 5 pmoleswd270 was added to a reaction containing [γ-³²P] ATP (3 μl of 3,000Ci/mmole at 10 mCi/ml in a 20 μl reaction volume), a 10X reaction buffer(700 mM Tris-HCl, pH 7.8, 100 mM MgCl₂, 50 mM DTT), and 10 units T4polynucleotide kinase (Promega Corporation, Madison, Wis.). The reactionwas incubated 20 minutes at 37 ° C. to allow the transfer of theradioactive phosphate to the 5′ end of the oligonucleotide, thus makingit useful as a hybridization probe.

The labeled probe was then incubated with the nitrocellulose filterovernight at 45° C. in 3X SSC, 0.1% SDS, 10X Denhardt's reagent (0.2%BSA, 0.2% polyvinylpyrrolidone, 0.2% ficoll), 0.2 mg/ml heparin.Following incubation the filter was washed in several changes of 3X SSC,0.1% SDS at 45° C. The filter was blotted dry and exposed to KodakX-OMAT AR X-ray film (Eastman Kodak Company, Rochester, N.Y.) overnightat −70° C. with a DuPont Cronex Lightning Plus screen.

The labeled probe was then incubated with the nitrocellulose filterwhich was then washed and exposed to X-ray film to obtain anautoradiogram.

Examination of the autoradiogram identified two distinct EcoRIrestriction fragments, of approximately 5.0 kb and 7.0 kb, thatspecifically hybridized to the labeled wd270 probe. This resultindicated that strain EG4096 either contained two closely related, oridentical, copies of the cryET29 gene, both of which hybridize to thewd270 oligonucleotide.

5.5 Example 5 Cloning of the cryET29 Gene of B. thuringiensis EG4096

To isolate the 5.0 and 7.0 kilobase (kb) EcoRI restriction fragmentscontaining the cryET29 gene, total genomic DNA was isolated from strainEG4096 as described in Example 4. The DNA was digested with EcoRI andelectrophoresed through a 0.8% agarose, 1X TBE gel, overnight at 2 V/cmof gel length. The gel was stained with Ethidium bromide so that thedigested DNA could be visualized when exposed to long-wave UV light. Gelslices containing DNA fragments of approximately 5.0 and 7.0 kb wereexcised from the gel with a razor blade and placed in separate dialysisbags containing a small volume (1 ml) of 10 mM Tris-HCl, pH 8.0, 1 mMEDTA (TE). The DNA fragments were eluted from the gel slices into the TEbuffer by placing the dialysis bags in a horizontal electrophoresisapparatus filled with 1X TBE and applying 100 V for 2 hr. This resultsin the DNA fragments migrating out of the gel slice into the TE buffer.The TE buffer containing the eluted fragments was then phenol:chloroformextracted and ethanol precipitated.

To create a library in E. coli of the two sets of size selected EcoRlrestriction fragments (approximately 5.0 and 7.0 kb), the fragments wereligated into the cloning vector pUC18 (Yanisch-Perron, et al., 1985).The plasmid DNA vector pUC18 can replicate at a high copy number in Ecoli and carries the gene for resistance to the antibiotic ampicillin,which may be used as a selectable marker. The two sets of fragments weremixed, in separate reactions, with EcoRI-digested pUC18 that had beentreated with bacterial alkaline phosphatase (GibcoBRL, Gaithersburg,Md.) to remove the 5′ phosphates from the digested plasmid to preventre-ligation of the vector to itself. T4 ligase and a ligation buffer(Promega Corporation, Madison, Wis.) were added to the reactioncontaining the digested pUC18 and the size-selected EcoRI fragments.These were incubated at room temperature for 1 hour to allow theinsertion and ligation of the EcoRI fragments into the pUC 18 vectorDNA.

The ligation mixtures described above were introduced, separately, intotransformation-competent E. coli DH5α™ cells (purchased from GibcoBRL,Gaithersburg, Md.) following procedures described by the manufacturer.The transformed E. coli cells were plated on LB agar plates containing50 μg/ml ampicillin and incubated overnight at 37° C. Bothtransformations yielded approximately 300 ampicillin-resistant coloniesindicating the presence of a recombinant plasmid in the cells of eachcolony.

To isolate the colonies harboring the cloned 5.0 and 7.0 kb EcoRIfragments that contain the cryET29 gene sequences the transformed E.coli colonies were first transferred to nitrocellulose filters. This wasaccomplished by simply placing a circular filter (Millipore HATF 085 25,Millipore Corp., Bedford, Mass.) directly on top of the LB-ampicillinagar plates containing the transformed colonies. When the filter isslowly peeled off of the plate the colonies stick to the filter givingan exact replica of the pattern of colonies from the original plate.Enough cells from each colony are left on the plate that 5 to 6 hr ofgrowth at 37° C. will restore the colonies. The plates are then storedat 4° C. until needed. The nitrocellulose filters with the transferredcolonies were then placed, colony-side up, on fresh LB-ampicillin agarplates and allowed to grow at 37° C. until they reached a size ofapproximately 1 mm in diameter.

To release the DNA from the recombinant E. coli cells onto thenitrocellulose filter the filters were placed, colony-side up, on 2sheets of Whatman 3 MM Chr paper (Whatman International LTD., Maidstone,England) soaked with 0.5 N NaOH, 1.5 M NaCl for 15 min. This treatmentlyses the cells and denatures the released DNA allowing it to stick tothe nitrocellulose filter. The filters were then neutralized by placingthe filters, colony-side up, on 2 sheets of Whatman paper soaked with 1M NH₄-acetate, 0.02 M NaOH for 10 min. The filters were then rinsed in3X SSC, air dried, and baked for 1 hr at 80° C. in a vacuum oven toprepare them for hybridization.

The NH₂-terminal oligonucleotide specific for the cryET29 gene, wd270,was labeled at the 5′ end with γ-³²P and T4 polynucleotide kinase asdescribed above. The labeled probe was added to the filters in 3X SSC,0.1% SDS, 10X Denhardt's reagent (0.2% BSA, 0.2% polyvinylpyrrolidone,0.2% ficoll), 0.2 mg/ml heparin and incubated overnight at 45° C. Theseconditions were chosen to allow hybridization of the labeledoligonucleotide to related sequences present on the nitrocellulose blotsof the transformed E. coli colonies. Following incubation the filterswere washed in several changes of 3X SSC, 0.1% SDS at 45° C. The filterswere blotted dry and exposed to Kodak X-OMAT AR x-ray film (EastmanKodak Company, Rochester, N.Y.) overnight at −70° C. with a DuPontCronex Lightning Plus screen.

Several colonies from each transformation (the 5.0 and 7.0 kb ligationmixes described above) hybridized to wd270. These colonies wereidentified by lining up the signals on the autoradiogram with thecolonies on the original transformation plates. The isolated colonieswere then grown in LB-ampicillin liquid medium from which the cellscould be harvested and recombinant plasmid prepared by the standardalkaline-lysis miniprep procedure (described in Maniatis et al., 1982).The isolated plasmids were digested with the restriction enzyme EcoRI todetermine if the cloned fragments of EG4096 DNA were of the expectedsize. All of the hybridizing plasmids from both the 5.0 kb and 7.0 kbconstructions had the expected size insert fragment. The DNA from theseplasmid digests were electrophoresed through an agarose gel andtransferred to nitrocellulose as described above. The blot was thenhybridized with the oligonucleotide, wd270, that had been radioactivelylabeled at the 5′ end with γ32P and T4 polynucleotide kinase. EcoRIfragments from two of the five plasmids containing 5.0 kb insertshybridized to the probe confirming the presence of the cryET29 gene onthose fragments. One of the plasmids with the 5.0 insert containing thecryET29 gene was designated pEG1298. EcoRI fragments from five of thesix plasmids containing 7.0 kb inserts hybridized to the probeconfirming the presence of the cryET29 gene on those fragments. One ofthe plasmids with the 7.0 kb insert containing the cryET29 gene wasdesignated pEG1299,

The E. coli strain containing pEG1298 has been designated EG 11513.EG11513 has been deposited with the Agricultural Research CultureCollection, Northern Regional Research Laboratory (NRRL) havingAccession No. NRRL B-21624. The E. coli strain containing pEG 1299 hasbeen designated EG 11514.

5.6 Example 6 Determination of the DNA Sequence of the cryET29 Gene

A partial DNA sequence of the genes cloned on pEG1298 and pEG1299 wasdetermined following established dideoxy chain-termination DNAsequencing procedures (Sanger et al., 1977). Preparation of the doublestranded plasmid template DNA was accomplished using a Qiagen PlasmidKit (Qiagen Inc., Chatsworth, Calif.) following manufacturer'sprocedures. The sequencing reactions were performed using the Sequenase™Version 2.0 DNA Sequencing Kit (United States Biochemical/Amersham LifeScience Inc., Cleveland, Ohio) following manufacturer's procedures andusing ³⁵S-dATP as the labeling isotope (obtained from Du Pont NEN®Research Products, Boston, Mass.). Denaturing gel electrophoresis of thereactions was done on a 6% (w/v) acrylamide, 42% (w/v) urea sequencinggel. The dried gel was exposed to Kodak X-OMAT AR X-ray film (EastmanKodak Company, Rochester, N.Y.) overnight at room temperature.

The NH₂-terminal specific oligonucleotide wd270 was used as the initialsequencing primer. The partial DNA sequences indicated that the plasmidspEG1298 and pEG1299 contained either identical, or nearly identical,copies of the cryET29 gene of B. thuringiensis strain EG4096. The entireDNA sequence for the copies of cryET29 on the two plasmids was completedusing the procedures described above. Successive oligonucleotides to beused for priming sequencing reactions were designed from the sequencingdata of the previous set of reactions. In this way the DNA sequencingprogressed along both the top and bottom strand of the cryET29 gene in astep-wise fashion.

The DNA sequence of both copies of the cryET29 (SEQ ID NO: 1) gene isidentical and is shown in FIG. 1. The protein coding portion of thecryET29 gene is comprised of 696 nucleotides, including a stop codon.The CryET29 protein (SEQ ID NO:2), as deduced from the DNA sequence,consists of 231 amino acids with a predicted molecular mass of 26,194daltons.

Database searches were then conducted to determine if the deduced aminoacid sequence of the CryET29 protein shares identity with othercharacterized proteins, especially other insecticidal toxin proteins.Database searches using on-line servers were performed with the BLASTPprogram (Altschul et al., 1990) provided by the National Center forBiotechnology Information (Bethesda, Md.). The BLASTP searches were runwith the BLOSUM62 matrix. The searched database consisted ofnon-redundant GenBank CDS translations+PDB+SwissProt+SPupdate+PIR.

Only four proteins in these databases were identified with anysignificant identity to CryET29. These included the dipteran toxin CytB(55% identity; Koni and Ellar, 1993); the coleopteran/dipteran toxinCytA (44.2% identity; Ward et al., 1984); the dipteran toxin PS201T6(41.1% identity; Intl. Pat. Appl. Publ. No. WO 95/02693) and the 27-kDaBacillus thuringiensis morrissoni dipteran toxin (44.2% identity; Earpand Ellar, 1987).

5.7 Example 7 Expression of the Cloned cryET29 Gene

To characterize the properties of the CryET29 protein it was necessaryto express the cloned cryET29 gene in B. thuringiensis cells that arenegative for crystal proteins (Cry⁻). The cloned EcoRI fragments onpEG1298 and pEG1299 was inserted into a plasmid vector capable ofreplicating in B. thuringiensis, thus allowing the expression of clonedgenes.

pEG1298 and pEG1299 were digested with EcoRI to remove the cloned 5 kband 7 kb fragments, respectively. The digested plasmids were resolved onan agarose gel and the desired fragments were purified from gel slicesusing the GeneClean® procedure of Bio101, Inc. (Vista, Calif.). Thefragments were ligated, separately, into a B. thuringiensis/E. colishuttle vector that had been digested with EcoRI and treated withbacterial alkaline phosphatase. The shuttle vector pEG1297 had beenconstructed by ligating the 3.1 kb EcoRI fragment of the Bacillus pNN101(Norton et al., 1985) into NdeI digested pUC18. pEG1297 is capable ofreplication in both E. coli and B. thuringiensis and confers Amp^(R) toE. coli and tetracycline (Tet) resistance (Tet^(R)) to B. thuringiensis.The two ligation mixtures were first introduced into E. coli DH5α™ cellsby transformation procedures described by the manufacturers (Gibco-BRL,Gaithersburg, Md.). Plasmid DNA was prepared from Amp^(R) transformantsand restriction enzyme analysis was performed to confirm the properconstruction. The plasmid consisting of the 5-kb EcoRI fragment ofpEG1298 inserted into pEG1297 was designated pEG1302. The plasmidconsisting of the 7-kb EcoRI fragment of pEG1299 inserted into pEG1297was designated pEG1303.

pEG1302 and pEG1303 were separately introduced into a Cry⁻ B.thuringiensis strain, EG10368, by electroporation (Macaluso and Mettus,1991). Cells transformed to tetracycline resistance were selected byincubation overnight on LB agar plates containing 10 μg/ml Tet. OneTet^(R) colony from each transformation was selected for furtheranalysis. Recombinant strain EG11494 contains pEG1302 (NRRL B-21583) andrecombinant strain EG 11502 contains pEG1303.

EG11494 and EG11502 were grown in C2 sporulation medium containing 10μg/ml tetracycline for 3 days at 30° C. until sporulation and cell lysishad occurred. Microscopic examination of the sporulated culturesdemonstrated that the recombinant strains were producing smallcrystalline inclusions. These crystals resemble the crystals produced bythe wild-type strain EG4096, indicating that the cryET29 gene in eachrecombinant was a functional gene capable of directing the expression ofthe CryET29 protein.

The sporulated cultures of EG 11494 and EG 11502 were harvested bycentrifugation, washed, and resuspended in 0.005% Triton X-100® inone-tenth the original volume. The crystal protein in the suspensionswas characterized by SDS-PAGE analysis which revealed the production ofan approximately 25-kDa protein by both EG11494 and EG11502. The 25-kDaproteins produced by the recombinant strains are identical in size asdetermined by migration on an SDS gel, to the crystal protein of EG4096.

The amount of toxin protein contained in a particular sample wasquantified for insect bioassays by SDS-PAGE. The Coomassie stainedSDS-PAGE gel was scanned on a densitometer and compared with a standardcurve generated by loading known amounts of a protein, such as bovineserum albumin, on the same gel.

5.8 Example 8 Toxicity of CryET29 to Southern Corn Rootworm Larvae

The toxicity to southern corn rootworm (SCRW) larvae (Diabroticaundecimpunctata howardi) was determined for wild-type B. thuringiensisEG4096 and for the two recombinant strains expressing the CryET29protein, EG11494 and EG11502.

EG4096, EG11494, and EG11502 were grown in C2 medium at 30° C. for 3days until sporulation and cell lysis had occurred. The cultures wereharvested by centrifugation, washed twice in 1X original volume 0.005%Triton X-100®, and resuspended in 1/10 the original culture volume on0.005% Triton X-100®. For comparison, a recombinant B. thuringiensisstrain, EG 11535, expressing the coleopteran-toxic protein CryIIIB2(Donovan et al., 1992), was grown and harvested in the same manner.

SCRW larvae were bioassayed via surface contamination of an artificialdiet similar to Marrone et al. (1985), but without formalin. Eachbioassay consisted of eight serial aqueous dilutions with aliquotsapplied to the surface of the diet. After the diluent (an aqueous 0.005%Triton X-100® solution) had dried, first instar larvae were placed onthe diet and incubated at 28° C. Thirty-two larvae were tested per dose.Mortality was scored after 7 days. Data from replicated bioassays werepooled for probit analysis (Daum, 1970) with mortality being correctedfor control death, the control being diluent only (Abbot, 1925). Resultsare reported as the amount of CryET29 crystal protein per well (175 mm²of diet surface) resulting in an LC₅₀, the concentration killing 50% ofthe test insects. 95% confidence intervals are also reported (Table 5).TABLE 5 Insecticidal Activity of the CryET29 Protein to SCRW LarvaeSample LC₅₀ (μg protein/well) 95% C.I. EG4096 35.3 29-43 EG11494 24.320-30 EG11502 26.7 22-32 EG11535 (CryIIIB2) 17.8 14-23

The results shown in Table 5 demonstrate that the CryET29 protein hassignificant activity on larvae of the southern corn rootworm. TheCryET29 produced by the two recombinant strains, EG11494 and EG11502,also exhibit significant toxicity. The SCRW activity of the CryET29protein produced in EG 11494 and EG11502 is somewhat lower than thatseen for the CryIIIB2 protein, although the 95% confidence intervals dooverlap slightly, indicating that the difference may not be significant.

5.9 Example 9 Toxicity of CryET29 to Western Corn Rootworm Larvae

The toxicity to western corn rootworm (WCRW) larvae (Diabroticavirgifera virgifera) was determined for wild-type B. thuringiensisEG4096 and for the two recombinant strains expressing the CryET29protein, EG11494 and EG11502.

The samples were prepared and the bioassays performed essentially asdescribed for the SCRW assays in Example 8. The wild-type B.thuringiensis strain EG4961, which produces the Coleopteran-activeCryIIIB2 protein, was included in the assay as a positive control (Table6). TABLE 6 Insecticidal Activity of the CryET29 Protein to SCRW LarvaeSample LC₅₀ (μg protein/well) 95% C.I. EG4961 (CryIIIB2) 73.8 44-211EG4096 12.9  7-110 EG11494 8.7 4-19 EG11502 13.9 9-29

The results in Table 6 demonstrate that the CryET29 protein hassignificant activity on larvae of the WCRW. Furthermore, the activity ofthe CryET29 produced by the recombinant strains EG11494 and EG11502 havesignificantly higher activity (i.e., lower LC₅₀s) than the proteinproduced by the coleopteran-active B. thuringiensis strain EG4096961.

5.10 Example 10 Toxicity of CryET29 to Colorado Potato Beetle Larvae

The toxicity to Colorado potato beetle (CPB) (Leptinotarsa decemlineata)larvae was determined for the wild-type B. thuringiensis strain EG4096and for the recombinant strain expressing the CryET29 protein, EG11494.The recombinant strain EG7231, which expresses the CryIIIB2 protein, wasgrown for purposes of comparison.

The assay on CPB larvae was performed using similar techniques to theSCRW assay, except for the substitution of BioServe's #9380 insect diet(with potato flakes added) for the artificial diet. Mortality was scoredat three days instead of seven days. For this assay 16 insects were usedper dose (Table 7). TABLE 7 Percent Mortality of CPB Larvae Treated WithCryET29-Producing Strains Dose in μg/well EG4096 EG11494 EG7231(CryIIIB2) 4.375 100 68.75 8.75 100 75 9.375 100 17.5 100 75 35 100 93

The results shown in Table 7 demonstrate the insecticidal activity ofthe CryET29 protein on CPB larvae.

5.11 Example 11 Toxicity of B. thuringiensis EG4096 to Red Flour BeetleLarvae

Toxicity of EG4096 to red flour beetle larvae (Tribolium castaneum) wasdetermined by applying a washed and concentrated sporulated culture ofEG4096 to an artificial diet and allowing the larvae to feed on thediet. Sixteen larvae were treated in this manner and the percentmortality was scored after two weeks. Larvae treated with the EG4096suspension exhibited 44% mortality compared to 13% for the untreatedcheck. In addition the surviving larvae treated with EG4096 exhibitedsignificant stunting in their growth which is indicative of a sublethaldose of an active toxin. The larvae in the untreated check showed nosuch stunting. These results demonstrate that EG4096, which produces theCryET29 protein, is toxic to red flour beetle.

5.12 Example 12 Toxicity of B. thuringiensis EG4096 to Japanese BeetleLarvae

The toxicity to Japanese beetle (JB) larvae (Popillia japonica) wasdetermined for B. thuringiensis EG4096, which produces the CryET29protein. Freeze-dried powders were prepared from washed and concentratedsporulated cultures of EG4096. The amount of CryET29 protein present inthe sample was determined by SDS-PAGE and quantitative densitometry ofthe Coomassie stained gels.

The freeze-dried powders were resuspended in a diluent containing 0.005%Triton X-100® and incorporated into 100 ml of hot (50-60° C.) liquidartificial diet (based on the insect diet described by Ladd (1986). Themixtures were allowed to solidify in Petri dishes, and 19-mm diameterplugs of the solidified diet were placed into ⅝ ounce plastic cups. OneJB larva was introduced per cup which were then covered with a lid andheld at 25° C. for fourteen days before larval mortality was scored.

Table 8 shows the average of results from two replications of thebioassay using 20 larvae per replication. The dosages were based on theamount of CryET29 protein in the sample. Percent mortality was correctedaccording to Abbott (1925). TABLE 8 Toxicity of EG4096 to JapaneseBeetle Larvae Amount CryET29 (ppm) % Mortality  250 ppm 9  500 ppm 691000 ppm 92 2000 ppm 96

The results shown in Table 8 demonstrate that the CryET29 proteinproduced by EG4096 has significant insecticidal activity on JB larvae.

5.13 Example 13 Toxicity of B. thuringiensis EG4096 to Cat Flea Larvae

The toxicity to larvae of the cat flea (Ctenocephalides felis) wasdetermined for B. thuringiensis EG4096, which produces the CryET29protein. Freeze-dried powders were prepared from washed and concentratedsporulated cultures of EG4096. The amount of CryET29 protein present inthe sample was determined by SDS-PAGE.

To perform the bioassay an amount of the freeze-dried powder containing1 mg of CryET29 protein was mixed with 1 gram of dried bovine bloodresulting in a concentration of 1000 ppm. The mixture was suspended in0.1% Triton X-100® and poured into a glass Petri dish to dry. The driedsample was then ground into a fine powder and evenly distributed into 32bioassay wells. One cat flea larva was added to each well which was thencovered with a lid and kept at high humidity. The assays were thenscored after seven days.

The assay is performed in this manner using a powder of EG4096 as thesample and the results are shown in Table 9. Thirty-two larvae wereassayed at each dose. Percent mortality was scored after 1, 4, and 7days. A B. thuringiensis strain that does not produce a toxin protein,EG2205, was used to assess control mortality. TABLE 9 Toxicity of EG4096to First Instar Cat Flea Larvae % Mortality Strain CryET29 (ppm) 1 Day 4Day 7 Day EG4096 500 6.25 15.60 15.60 EG4096 1000 9.40 34.40 43.75EG4096 5000 46.90 78.10 87.50 EG4096 10000 84.40 93.75 100.00 EG2205 Notoxin 3.10 15.60 15.60

The results shown in Table 9 demonstrate that the CryET29 proteinproduced by Bacillus thuringiensis strain EG4096 has significantinsecticidal activity on larvae of the cat flea, Ctenocephalides felis.

The uniqueness of the activity of the CryET29 toxin on cat fleas larvaewas demonstrated by assaying other Bacillus thuringiensis insecticidalcrystal proteins in the manner described above. Samples containingspores and crystals were tested from recombinant strains of B.thuringiensis expressing the following toxin proteins: Cry1Aa, Cry1Ab,Cry1Ac, Cry2S, Cry3A, Cry3B, Cry3B2, and Cry3B3. The characteristics ofthese other classes of insecticidal crystal protein genes are describedby Hofte et al., (1989). For a detailed description of the Cry3 toxins,see U.S. Pat. No. 5,187,091 and U.S. Patent No. 5,264,364, specificallyincorporated herein by reference. None of these toxins showed anytoxicity toward the larvae of the cat flea indicating that the CryET29toxin protein is unique among B. thuringiensis insecticidal proteinsisolated to date with respect to its cat flea larvae toxicity.

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The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-70. (canceled)
 71. An isolated nucleic acid segment characterized as:(a) a nucleic acid segment consisting of at least 14 nucleotides thathas the same sequence as, or is complementary to, 14 contiguousnucleotides of SEQ ID NO: 1; or (b) a nucleic acid segment consisting ofat least 14 nucleotides that specifically hybridizes to SEQ ID NO: 1 ora complement thereof.
 72. The isolated nucleic acid segment of claim 71consisting of a sequence as set forth in SEQ ID NO:4.
 73. A method fordetecting a nucleic acid sequence encoding a Coleopteran toxic crystalprotein, comprising the steps of: (a) obtaining sample nucleic acidssuspected of encoding a CryET29 crystal protein; (b) contacting saidsample nucleic acids with the isolated nucleic acid segment of claim 71or 72 under conditions effective to allow hybridization of complementarynucleic acids; and (c) detecting the hybridized complementary nucleicacids thus formed.
 74. The method of claim 73, wherein said nucleic acidsegment comprises a detectable label and said hybridized complementarynucleic acids are detected by detecting said label.
 75. A nucleic aciddetection kit comprising, in suitable container means, the nucleic acidsegment of claim 71 or 72 and a detection reagent.
 76. A recombinantvector comprising a nucleic acid sequence encoding a CryET29 protein asset forth in SEQ ID NO:2 or a peptide having an amino acid sequenceessentially as set forth in SEQ ID NO:2.
 77. The recombinant vector ofclaim 76 comprising a nucleic acid sequence as set forth in SEQ ID NO: 1or a nucleic acid sequence encoding said peptide.
 78. The recombinantvector of claim 76 or 77, wherein said nucleic acid sequence is operablylinked to a promoter.
 79. The recombinant vector of claim 76 or 77,further defined as recombinant vector pEG1298, pEG1299, pEG1302 orpEG1303.
 80. An artificial diet for Coleopteran or Ctenocephalidesinsects, comprising a CryET29 protein as set forth in SEQ ID NO:2. 81.The artificial diet of claim 80, comprising from about 1% to about 50%by weight of said CryET29 protein.
 82. The artificial diet of claim 80or 81, wherein said CryET29 protein is prepared by a process comprisingthe steps of: a) culturing a Bacillus thuringiensis NRRL B-21582, NRRLB-21583 or Escherichia coli NRRL B-21624 cell under conditions effectiveto produce a CryET29 protein; and b) obtaining said CryET29 protein fromsaid cell.
 83. The artificial diet of claim 82, wherein step b) furthercomprises obtaining said CryET29 protein in an amount from between about1% to about 50% by weight.
 84. The artificial diet of claim 80, whereinsaid Coleopteran insects include southern corn rootworm (Diabroticaundecimpunctata howardi), western corn rootworm (Diabrotica virgiferavirgifera), Colorado potato beetle (Leptinotarsa decemlineata), redflour beetle (Tribolium castaneum), and Japanese beetle (Popilliajaponica).
 85. The artificial diet of claim 80, wherein saidCtenocephalides insects include cat flea (Ctenocephalides felis) and dogflea (Ctenocephalides canis).
 86. A method for killing Coleopteran orCtenocephalides insects, comprising the step of providing the artificialdiet of claim 80 to Coleopteran or Ctenocephalides insect larvae.